Immunosuppressant Target Proteins

ABSTRACT

The present invention relates to the discovery of novel proteins of mammalian origin which are immediate downstream targets for FKBP/rapamycin complexes.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/250,795,filed May 27, 1994 and entitled “Immunosuppressant Target Proteins”, thespecification of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

Cyclosporin A, FK506, and rapamycin are microbial products with potentimmunosuppressive properties that result primarily from a selectiveinhibition of T lymphocyte activation. Rapamycin was first described asan antifungal antibiotic extracted from a streptomycete (Streptomyceshygroscopicus) (Vezina et al. (1975). J. Antibiot., 28:721; Sehgal etal. (1975) J. Antibiot. 28:727; and Sehgal et al., U.S. Pat. No.3,929,992). Subsequently, the macrolide drug rapamycin was shown toexhibit immunosuppressive as well as antineoplastic andantiproliferative properties (Morris (1992) Transplant Res 6:39-87).

Each of these compounds, cyclosporin A, FK506 and rapamycin, suppressthe immune system by blocking distinctly different biochemical reactionswhich would ordinarily initiate the activation of immune cells. Briefly,cyclosporin A and FK506 act soon after Ca²⁺-dependent T-cell activationto prevent the synthesis of cytokines important for the perpetuation andamplification of the immune response. Rapamycin acts later to blockmultiple affects of cytokines on immune cells including the inhibitionof interleukin-2 (IL2)-triggered T-cell proliferation, but itsantiproliferative effects are not restricted solely to T and B cells.Rapamycin also selectively inhibits the proliferation of growthfactor-dependent and growth factor-independent nonimmune cells.Rapamycin is generally believed to inhibit cell proliferation byblocking specific signaling events necessary for the initiation of Sphase in a number of cell types, including lymphocytes (Bierer et al.(1990) PNAS 87:9231-9235; and Dumont et al. (1990) J. Immunol144:1418-1424), as well as non-immune cells, such as hepatocytes(Francavilla et al. (1992) Hepatology 15:871-877; and Price et al.(1992) Science 257:973-977). Several lines of evidence suggest that theassociation of rapamycin with different members of a family ofintracellular FK506/rapamycin binding proteins (FKBPs) is necessary forthe inhibition of G₁ progression as mediated by rapamycin. For instance,the actions of rapamycin are reversed by an excess of the structurallyFKBP-ligands FK506 or 506BD (Bierer et al. supra.; Dumont et al. supra.;and Bierer et al. (1990) Science 250:556-559).

Cyclosporin A binds to a class of proteins called cyclophilins (Walsh etal. (1992) J. Biol. Chem. 267:13115-13118), whereas the primary targetsfor both FK506 and rapamycin, as indicated above, are the FKBPs (Hardinget al. (1989) Nature 341:758-7601; Siekienka et al. (1989) Nature341:755-757; and Soltoff et al. (1992 J. Biol. Chem. 267:17472-17477).Both the cyclophilin/cyclosporin and FKBPI2/FK506 complexes bind to aspecific protein phosphatase (calcineurin) which is hypothesized tocontrol the activity of IL-2 gene specific transcriptional activators(reviewed in Schreiber (1991) Cell 70:365-368). In contrast, thedownstream cellular targets for the rapamycin-sensitive signalingpathway have not been especially well characterized, particularly withregard to the identity of the direct target of the FKBP-rapamycincomplex.

The TOR1 and TOR2 genes of S. cerevisiae were originally identified bymutations that rendered cells resistant to rapamycin (Heitman et al.(1991) Science 253:905-909) and there was early speculation that theFKBP/rapamycin complex might inhibit the cellular function of the TORgene product by binding directly to a phosphoserine residue of eitherTOR1 or TOR2. Subsequently, however, new models for rapamycin druginteraction have been proposed which do not involve direct binding ofthe FKBP/rapamycin complex to the TOR proteins. For example, based onexperimental data regarding cyclin-cdk activity in rapamycin treatedcells, Stuart Schreiber wrote in Albers et al. (1993) J. Biol. Chem.268:22825-22829:

-   -   “Although it is possible the TOR2 gene product is a direct        target of the FKBP-rapamycin complex, a more likely explanation        is that the TOR2 gene product lies downstream of the direct        target of rapamycin and that the TOR2 mutation caused the        protein to be constitutively active. If the latter model is        correct, then the TOR2 gene product joins p70^(s6k),        cyclin-dependent kinases, and cyclin D1 as proteins that lie        downstream of the direct target of the FKBP-rapamycin complex        and have been shown to play important roles in cell cycle        progression. The identification of the direct target of the        FKBP-rapamycin complex will likely reveal an upstream component        of the signal transduction pathway that leads to G1 progression        and will help delineate the signal transduction pathways that        link growth factor-mediated signaling events and cyclin-cdk        activity required for cell cycle progression.”

Likewise, after studying the role of TOR1 and TOR2 mutations inrapamycin-resistant yeast cells, George Livi wrote in Cafferkey et al.(1993) Mol. Cell. Biol. 13:6012-6023:

-   -   “Thus, the amino acid changes that we have identified in the        rapamycin-released DRR1 [TOR1] protein may allow it to        compensate for the loss of the proliferative signal inhibited by        rapamycin by constitutively activating an alternative signal        rather than by preventing its association with the        FKBP12-rapamycin complex. The positions of the mutations within        the kinase domain, but in a region not shared by the PI        3-kinases, support this idea. Therefore, it is entirely possible        that DRR1 is not a component of the rapamycin-sensitive pathway        in wild-type yeast cells. Instead, missense mutations in DRR1 at        Ser-1972 may alter its normal activity and allow it to        substitute for the function of an essential protein which is the        true target of rapamycin.”

It is an object of the present invention to identify cellular proteinswhich are the direct downstream target proteins for the FKBP/rapamycincomplex, and isolate the genes encoding those proteins.

SUMMARY OF THE INVENTION

The present invention relates to the discovery of novel proteins ofmammalian origin which are immediate downstream targets forFKBP/rapamycin complexes. As described herein, a drug-dependentinteraction trap assay was used to isolate a number of proteins whichinteract with an FK506-binding protein/rapamycin complex, and which arecollectively referred to herein as “RAP-binding proteins” or “RAP-BPs”.In particular, mouse and human genes have been cloned for a protein(referred to herein as “RAPT1”) which is apparently related to the yeastTOR1 and TOR2 gene products. Furthermore, a novel ubiquitin-conjugatingenzyme (referred to herein as “rap-UBC”) has been cloned based on itsability to bind FKBP/rapamycin complexes. In addition, a RAPT1-likeprotein was cloned from the human pathogen Candida albicans. The presentinvention, therefore, makes available novel proteins (both recombinantand purified forms), recombinant genes, antibodies to RAP-bindingproteins, and other novel reagents and assays for diagnostic andtherapeutic use.

The present invention relates to the discovery in eukaryotic cells,particularly human cells, of novel protein-protein interactions betweenthe Wilms tumor regulatory protein rapamycin complexes and certaincellular proteins, referred to hereinafter as “RAP-binding proteins” or“RAP-BP”.

In general, the invention features a mammalian RAPT1 polypeptide,preferably a substantially pure preparation of a RAPT1 polypeptide, or arecombinant RAPT1 polypeptide. In preferred embodiments the polypeptidehas a biological activity associated with its binding to rapamycin,e.g., it remains the ability to bind to an FKBP/rapamycin complex,though it may be able to either agonize or antagonize assembly ofrapamycin-dependent complexes. The polypeptide can be identical to apolypeptide shown in one of SEQ ID No: 2 or 12, or it can merely behomologous to that sequence. For instance, the polypeptide preferablyhas an amino acid sequence at least 60% homologous to the amino acidsequence of at least one of either SEQ ID No: 2 or 12, though highersequence homologies of, for example, 80%, 90% or 95% are alsocontemplated. The polypeptide can comprise the full length protein, or aportion of a full length protein, such as the RAPT1 polypeptidesrepresented in either SEQ ID No: 2 or 12, or an even smaller fragment ofthat protein, which fragment may be, for instance, at least 5, 10, 20,50 or 100 amino acids in length. As described below, the RAPT1polypeptide can be either an agonist (e.g. mimics), or alternatively, anantagonist of a biological activity of a naturally occurring form of theprotein, e.g., the polypeptide is able to modulate assembly of rapamycincomplexes, such as complexes involving FK506-binding proteins, or cellcycle regulatory proteins.

In a preferred embodiment, a peptide having at least one biologicalactivity of the subject RAPT1 polypeptides may differ in amino acidsequence from the sequence in SEQ ID No: 2 or 12, but such differencesresult in a modified protein which functions in the same or similarmanner as the native RAPT1 protein or which has the same or similarcharacteristics of the native RAPT1 protein. However, homologs of thenaturally occurring protein are contemplated which are antagonistic ofthe normal cellular role of the naturally occurring protein.

In yet other preferred embodiments, the RAPT1 protein is a recombinantfusion protein which includes a second polypeptide portion, e.g., asecond polypeptide having an amino acid sequence unrelated to the RAPT1polypeptide portion, e.g. the second polypeptide portion isglutathione-S-transferase, e.g. the second polypeptide portion is a DNAbinding domain of transcriptional regulatory protein, e.g. the secondpolypeptide portion is an RNA polymerase activating domain, e.g. thefusion protein is functional in a two-hybrid assay.

Yet another aspect of the present invention concerns an immunogencomprising a RAPT1 peptide in an immunogenic preparation, the immunogenbeing capable of eliciting an immune response specific for the RAPT1polypeptide; e.g. a humoral response, e.g. an antibody response; e.g. acellular response. In preferred embodiments, the immunogen comprising anantigenic determinant, e.g. a unique determinant, from a proteinrepresented by SEQ ID No: 2 and/or 12.

A still further aspect of the present invention features an antibodypreparation specifically reactive with an epitope of the RAPT1immunogen.

In another aspect, the invention features a ubiquitin conjugating enzyme(rap-UBC), preferably a substantially pure preparation of a rap-UBCpolypeptide, or a recombinant rap-UBC polypeptide. As above, inpreferred embodiments the rap-UBC polypeptide has a biological activityassociated with its binding to rapamycin, e.g., it retains the abilityto bind to a rapamycin complex, and may additionally retain a ubiquitinconjugating activity. The polypeptide can be identical to thepolypeptide shown in SEQ ID No: 24, or it can merely be homologous tothat sequence. For instance, the polypeptide preferably has an aminoacid sequence at least 60% homologous to the amino acid sequence in SEQID No: 24, though higher sequence homologies of, for example, 80%, 90%or 95% are also contemplated. The rap-UBC polypeptide can comprise thefull length polypeptide represented in SEQ ID No: 24, or it can comprisea fragment of that protein, which fragment may be, for instance, atleast 5, 10, 20, 50 or 100 amino acids in length. The rap-UBCpolypeptide can be either an agonist (e.g. mimics), or alternatively, anantagonist of a biological activity of a naturally occurring form of theprotein.

In a preferred embodiment, a peptide having at least one biologicalactivity of the subject rap-UBC polypeptide may differ in amino acidsequence from the sequence in SEQ ID No: 24, but such differences resultin a modified protein which functions in the same or similar manner asthe native rap-UBC or which has the same or similar characteristics ofthe native protein. However, homologs of the naturally occurring rap-UBCprotein are contemplated which are antagonistic of the normal cellularrole of the naturally occurring protein.

In yet other preferred embodiments, the rap-UBC protein is a recombinantfusion protein which includes a second polypeptide portion, e.g., asecond polypeptide having an amino acid sequence unrelated to therap-UBC sequence, e.g. the second polypeptide portion isglutathione-S-transferase, e.g. the second polypeptide portion is a DNAbinding domain of transcriptional regulatory protein, e.g. the secondpolypeptide portion is an RNA polymerase activating domain, e.g. thefusion protein is functional in a two-hybrid assay.

Yet another aspect of the present invention concerns an immunogencomprising a rap-UBC peptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for therap-UBC polypeptide; e.g. a humoral response, e.g. an antibody response;e.g. a cellular response. In preferred embodiments, the immunogencomprising an antigenic determinant, e.g. a unique determinant, from aprotein represented by SEQ ID No: 24.

A still further aspect of the present invention features an antibodypreparation specifically reactive with an epitope of the rap-UBCimmunogen.

In still another aspect, the invention features a RAPT1-like polypeptidefrom a Candida species (caRAPT1), preferably a substantially purepreparation of a caRAPT1 polypeptide, or a recombinant caRAPT1polypeptide. As above, in preferred embodiments the caRAPT1 polypeptidehas a biological activity associated with its binding to rapamycin,e.g., it retains the ability to bind to a rapamycin complex, such as anFKBP/rapamycin complex. The polypeptide can be identical to thepolypeptide shown in SEQ ID No: 14, or it can merely be homologous tothat sequence. For instance, the caRAPT1 polypeptide preferably has anamino acid sequence at least 60% homologous to the amino acid sequencein SEQ ID No: 14, though higher sequence homologies of, for example,80%, 90% or 95% are also contemplated. The caRAPT1 polypeptide cancomprise the entire polypeptide represented in SEQ ID No: 14, or it cancomprise a fragment of that protein, which fragment may be, forinstance, at least 5, 10, 20, 50 or 100 amino acids in length. ThecaRAPT1 polypeptide can be either an agonist (e.g. mimics), oralternatively, an antagonist of a biological activity of a naturallyoccurring form of the protein.

In a preferred embodiment, a peptide having at least one biologicalactivity of the subject caRAPT1 polypeptide may differ in amino acidsequence from the sequence in SEQ ID No: 14, but such differences resultin a modified protein which functions in the same or similar mariner asthe native caRAPT1 or which has the same or similar characteristics ofthe native protein. However, homologs of the naturally occurring caRAPT1protein are contemplated which are antagonistic of the normal cellularrole of the naturally occurring protein.

In yet other preferred embodiments, the caRAPT1 protein is a recombinantfusion protein which includes a second polypeptide portion, e.g., asecond polypeptide having an amino acid sequence unrelated to thecaRAPT1 sequence, e.g. the second polypeptide portion isglutathione-S-transferase, e.g. the second polypeptide portion is a DNAbinding domain of transcriptional regulatory protein, e.g. the secondpolypeptide portion is an RNA polymerase activating domain, e.g. thefusion protein is functional in a two-hybrid assay.

Yet another aspect of the present invention concerns an immunogencomprising a caRAPT1 peptide in an immunogenic preparation, theimmunogen being capable of eliciting an immune response specific for thecaRAPT1 polypeptide; e.g. a humoral response, e.g. an antibody response;e.g. a cellular response. In preferred embodiments, the immunogencomprising an antigenic determinant, e.g. a unique determinant, from aprotein represented by SEQ ID No: 14.

A still further aspect of the present invention features an antibodypreparation specifically reactive with an epitope of the caRAPT1immunogen.

Another aspect of the present invention provides a substantiallyisolated nucleic acid having a nucleotide sequence which encodes a RAPT1polypeptide. In preferred embodiments: the encoded polypeptidespecifically binds a rapamycin complexes and/or is able to eitheragonize or antagonize assembly of rapamycin-containing proteincomplexes. The coding sequence of the nucleic acid can comprise aRAPT1-encoding sequence which can be identical to the cDNA shown in SEQID No: 1 or 11, or it can merely be homologous to that sequence. Forinstance, the RAPT1-encoding sequence preferably has a sequence at least60% homologous to one or both of the nucleotide sequences in SEQ ID No:1 or 11, though higher sequence homologies of, for example, 80%, 90% or95% are also contemplated. The nucleic acid can comprise the nucleotidesequence represented in SEQ ID No: 1, or it can comprise a fragment ofthat nucleic acid, which fragment may be, for instance, encode afragment of which is, for example, at least 5, 10, 20, 50, 100 or 133amino acids in length. The polypeptide encoded by the nucleic acid canbe either an agonist (e.g. mimics), or alternatively, an antagonist of abiological activity of a naturally occurring form of the RAPT1 protein,e.g., the polypeptide is able to modulate rapamycin-mediated proteincomplexes.

Furthermore, in certain preferred embodiments, the subject RAPT1 nucleicacid will include a transcriptional regulatory sequence, e.g. at leastone of a transcriptional promoter or transcriptional enhancer sequence,which regulatory sequence is operably linked to the RAPT1 gene sequence.Such regulatory sequences can be used in to render the RAPT1 genesequence suitable for use as an expression vector.

In yet a further preferred embodiment, the nucleic acid hybridizes understringent conditions to a nucleic acid probe corresponding to at least12 consecutive nucleotides of SEQ ID No: 1 and/or 11; preferably to atleast 20 consecutive nucleotides, and more preferably to at least 40consecutive nucleotides.

Another aspect of the present invention provides a substantiallyisolated nucleic acid having a nucleotide sequence which encodes arap-UBC polypeptide. In preferred embodiments: the encoded polypeptidespecifically binds a rapamycin complexes and/or is able to eitheragonize or antagonize assembly of rapamycin-containing proteincomplexes. The coding sequence of the nucleic acid can comprise arap-UBC-encoding sequence which can be identical to the cDNA shown inSEQ ID No: 23, or it can merely be homologous to that sequence. Forinstance, the rap-UBC-encoding sequence preferably has a sequence atleast 60% homologous to the nucleotide sequences in SEQ ID No: 23,though higher sequence homologies of, for example, 80%, 90% or 95% arealso contemplated. The nucleic acid can comprise the nucleotide sequencerepresented in SEQ ID No: 23, or it can comprise a fragment of thatnucleic acid, which fragment may be, for instance, encode a fragment ofwhich is, for example, at least 5, 10, 20, 50, or 100 amino acids inlength. The polypeptide encoded by the nucleic acid can be either anagonist (e.g. mimics), or alternatively, an antagonist of a biologicalactivity of a naturally occurring form of the rap-UBC protein, e.g., thepolypeptide is able to modulate rapamycin-mediated protein complexes.

Furthermore, in certain preferred embodiments, the subject rap-UBCnucleic acid will include a transcriptional regulatory sequence, e.g. atleast one of a transcriptional promoter or transcriptional enhancersequence, which regulatory sequence is operably linked to the rap-UBCgene sequence. Such regulatory sequences can be used in to render therap-UBC gene sequence suitable for use as an expression vector.

In yet a further preferred embodiment, the nucleic acid hybridizes understringent conditions to a nucleic acid probe corresponding to at least12 consecutive nucleotides of SEQ ID No: 23; preferably to at least 20consecutive nucleotides, and more preferably to at least 40 consecutivenucleotides.

Another aspect of the present invention provides a substantiallyisolated nucleic acid having a nucleotide sequence which encodes acaRAPT1 polypeptide. In preferred embodiments: the encoded polypeptidespecifically binds a rapamycin complexes and/or is able to eitheragonize or antagonize assembly of rapamycin-containing proteincomplexes. The coding sequence of the nucleic acid can comprise acaRAPT1-encoding sequence which can be identical to the cDNA shown inSEQ ID No: 13, or it can merely be homologous to that sequence. Forinstance, the caRAPT1-encoding sequence preferably has a sequence atleast 60% homologous to the nucleotide sequences in SEQ ID No: 13,though higher sequence homologies of, for example, 80%, 90% or 95% arealso contemplated. The nucleic acid can comprise the nucleotide sequencerepresented in SEQ ID No: 13, or it can comprise a fragment of thatnucleic acid, which fragment may be, for instance, encode a fragment ofwhich is, for example, at least 5, 10, 20, 50, 100 or 133 amino acids inlength. The polypeptide encoded by the nucleic acid can be either anagonist (e.g. mimics), or alternatively, an antagonist of a biologicalactivity of a naturally occurring form of the caRAPT1 protein, e.g., thepolypeptide is able to modulate rapamycin-mediated protein complexes.

Furthermore, in certain preferred embodiments, the subject caRAPT1nucleic acid will include a transcriptional regulatory sequence, e.g. atleast one of a transcriptional promoter or transcriptional enhancersequence, which regulatory sequence is operably linked to the caRAPT1gene sequence. Such regulatory sequences can be used in to render thecaRAPT1 gene sequence suitable for use as an expression vector.

In yet a further preferred embodiment, the nucleic acid hybridizes understringent conditions to a nucleic acid probe corresponding to at least12 consecutive nucleotides of SEQ ID No: 13; preferably to at least 20consecutive nucleotides, and more preferably to at least 40 consecutivenucleotides.

The invention also features transgenic non-human animals, e.g. mice,rats, rabbits or pigs, having a transgene, e.g., animals which include(and preferably express) a heterologous form of one, of the RAP-BP genesdescribed herein, e.g. a gene derived from humans, or which misexpressan endogenous RAP-BP gene, e.g., an animal in which expression of one ormore of the subject RAP-binding proteins is disrupted. Such a transgenicanimal can serve as an animal model for studying cellular disorderscomprising mutated or mis-expressed RAP-BP alleles or for use in drugscreening.

The invention also provides a probe/primer comprising a substantiallypurified oligonucleotide, wherein the oligonucleotide comprises a regionof nucleotide sequence which hybridizes under stringent conditions to atleast 10 consecutive nucleotides of sense or antisense sequence of oneof SEQ ID Nos: 1, 11, 13 or 24, or naturally occurring mutants thereof.In preferred embodiments, the probe/primer further includes a labelgroup attached thereto and able to be detected. The label group can beselected, e.g., from a group consisting of radioisotopes, fluorescentcompounds, enzymes, and enzyme co-factors. Probes of the invention canbe used as a part of a diagnostic test kit for identifying transformedcells, such as for detecting in a sample of cells isolated from apatient, a level of a nucleic acid encoding one of the subjectRAP-binding proteins; e.g. measuring the RAP-BP mRNA level in a cell, ordetermining whether the genomic RAP-BP gene has been mutated or deleted.Preferably, the oligonucleotide is at least 10 nucleotides in length,though primers of 20, 30, 50, 100, or 150 nucleotides in length are alsocontemplated.

In yet another aspect, the invention provides assay systems forscreening test compounds for an molecules which induce an interactionbetween a RAP-binding protein and a rapamycin/protein complexes. Anexemplary method includes the steps of (i) combining a RAP-bindingprotein of the invention, an FK506-binding protein, and a test compound,e.g., under conditions wherein, but for the test compound, theFK506-binding protein and the RAP-binding protein are unable tointeract; and (ii) detecting the formation of a drug-dependent complexwhich includes the FK506-binding protein and the RAP-binding protein. Astatistically significant change, such as an increase, in the formationof the complex in the presence of a test compound (relative to what isseen in the absence of the test compound) is indicative of a modulation,e.g., induction, of the interaction between the FK506-binding proteinand the RAP-binding protein. Moreover, primary screens are provided inwhich the FK506-binding protein and the RAP-binding protein are combinedin a cell-free system and contacted with the test compound; i.e. thecell-free system is selected from a group consisting of a cell lysateand a reconstituted protein mixture. Alternatively, FK506-bindingprotein and the RAP-binding protein are simultaneously expressed in acell, and the cell is contacted with the test compound, e.g. as aninteraction trap assay (two hybrid assay).

The present invention also provides a method for treating an animalhaving unwanted cell growth characterized by a loss of wild-typefunction of one or more of the subject RAP-binding proteins, comprisingadministering a therapeutically effective amount of an agent able toinhibit the interaction of the RAP-binding protein with other cellularor viral proteins. In one embodiment, the method comprises administeringa nucleic acid construct encoding a polypeptides represented in one ofSEQ ID Nos: 2, 12 or 24, under conditions wherein the construct isincorporated by cells deficient in that RAP-binding protein, and underconditions wherein the recombinant gene is expressed, e.g. by genetherapy techniques. In other embodiments, the action of anaturally-occurring RAP-binding protein is antagonized by therapeuticexpression of a RAP-BP homolog which is an antagonist of, for example,assembly of rapamycin-mediated complexes, or by delivery of an antisensenucleic acid molecule which inhibits transcription and/or translation ofthe targeted RAP-BP gene.

Another aspect of the present invention provides a method of determiningif a subject, e.g. a human patient, is at risk for a disordercharacterized by unwanted cell proliferation. The method includesdetecting, in a tissue of the subject, the presence or absence of agenetic lesion characterized by at least one of (i) a mutation of a geneencoding a protein represented by one of SEQ ID Nos: 1, 11 or 13, or ahomolog thereof; (ii) the mis-expression of a gene encoding a proteinrepresented by one of SEQ ID Nos: 1, 11 or 13; or (iii) themis-incorporation of a RAP-binding protein in a regulatory proteincomplex, e.g. a rapamycin-containing complex. In preferred embodiments:detecting the genetic lesion includes ascertaining the existence of atleast one of: a deletion of one or more nucleotides from the RAP-BPgene; an addition of one or more nucleotides to the gene, ansubstitution of one or more nucleotides of the gene, a gross chromosomalrearrangement of the gene; an alteration in the level of a messenger RNAtranscript of the gene; the presence of a non-wild type splicing patternof a messenger RNA transcript of the gene; or a non-wild type level ofthe protein.

For example, detecting the genetic lesion can include (i) providing aprobe/primer including an oligonucleotide containing a region ofnucleotide sequence which hybridizes to a sense or antisense sequence ofone of SEQ ID Nos: 1, 11 or 23, or naturally occurring mutants thereofor 5′ or 3′ flanking sequences naturally associated with the RAP-BPgene; (ii) exposing the probe/primer to nucleic acid of the tissue; and(iii) detecting, by hybridization of the probe/primer to the nucleicacid, the presence or absence of the genetic lesion; e.g. whereindetecting the lesion comprises utilizing the probe/primer to determinethe nucleotide sequence of the RAP-BP gene and, optionally, of theflanking nucleic acid sequences. For instance, the probe/primer can beemployed in a polymerase chain reaction (PCR) or in a ligation chainreaction (LCR). In alternate embodiments, the level of the RAP-bindingprotein is detected in an immunoassay using an antibody which isspecifically immunoreactive with a protein represented by one of SEQ IDNos: 1, 11 or 23.

Another aspect of the present invention concerns a novel in vivo methodfor the isolation of genes encoding proteins which physically interactwith a “bait” protein/drug complex. The method relies on detecting thereconstitution of a transcriptional activator in the presence of thedrug, particularly wherein the drug is a non-peptidyl small organicmolecule (e.g. <2500K), e.g. a macrolide, e.g. rapamycin, FK506 orcyclosporin. In particular, the method makes use of chimeric genes whichexpress hybrid proteins. The first hybrid comprises the DNA-bindingdomain of a transcriptional activator fused to the bait protein. Thesecond hybrid protein contains a transcriptional activation domain fusedto a “fish” protein, e.g. a test protein derived from a cDNA library. Ifthe fish and bait proteins are able to interact in a drug-dependentmanner, they bring into close proximity the two domains of thetranscriptional activator. This proximity is sufficient to causetranscription of a reporter gene which is operably linked to atranscriptional regulatory site responsive to the transcriptionalactivator, and expression of the marker gene can be detected and used toscore for the interaction of the bait protein/drug complex with anotherprotein.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the map of the pACT vector used to clone the humanRAPT1 clone. The RAPT1-containing version of pACT, termed “pIC524” hasbeen deposited with the ATCC.

FIG. 2 illustrates the interaction of FKBP12 and hRAPT1(rapamycin-binding domain) as a function of rapamycin concentration.INteraction is detected as β-galactosidase activity. No interaction isdetected if FK506 is used in place of rapamycin, or if lex.da (a controlplasmid) replaces FKBP12.

FIG. 3 illustrates the relative strengths of interaction between pairsof FK506-binding proteins and rapamycin-binding domain (BD) fusions inthe presence of varying concentrations of rapamycin, measured byβ-galactosidase expression (see Example 8). The yeast reporter strainVBY567 was transformed with the indicated pairs of plasmids. LexADNA-binding domain fusions to human FKBP12, yeast FKBP12 and anunrelated sequence serving as negative control were used as “baits”. TheVP16 acidic activation domain fusions to human RAPT1 BD, human RAPT1 BDcontaining the serine to arginine substitution, yeast Tor1 BD, yeastTor2 BD (not shown) and Candida albicans RAPT1 BD were tested forinteraction against the bait fusions. Transformants containing each pairof plasmids were tested for β-galactosidase expression on mediacontaining the chromogenic substrate X-gal. Colonies were scored aseither white (open bars) or blue (solid bars) after growth at 30° C. for2 days. The levels of β-galactosidase expression were qualitativelyscored by the intensity of the blue color, ranging from 1 (light blue)to 4 (deep blue).

DETAILED DESCRIPTION OF THE INVENTION

Recent studies have provided some remarkable insights into the molecularbasis of eukaryotic cell cycle regulation. Passage of a mammalian cellthrough the cell cycle is regulated at a number of key control points.Among these are the points of entry into and exit from quiescence (G₀),the restriction point, the G₁/S transition, and the G₂/M transition (forreview, see Draetta (1990) Trends Biol Sci 15:378-383; and Sherr (1993)Cell 73:1059-1065). Ultimately, information from these check-pointcontrols is integrated through the regulated activity of a group ofrelated kinases, the cyclin-dependent kinases (CDKs). For example, theG₁-to-S phase transition is now understood to be timed precisely by thetransient assembly of multiprotein complexes involving the periodicinteraction of a multiplicity of cyclins and cyclin-dependent kinases.

To illustrate, stimulation of quiescent T lymphocytes by cell-boundantigens triggers a complex activation program resulting in cell cycleentry (G₀-to-G₁ transition) and the expression of high affinityinterleukin-2 (IL-2) receptors. The subsequent binding of IL-2 to itshigh affinity receptor drives the progression of activated T cellsthrough a late G₁-phase “restriction point” (Pardee (1989) Science246:603-608), after which the cells are committed to complete arelatively autonomous program of DNA replication and, ultimately,mitosis.

One important outcome of the information concerning eukaryotic cellcycle regulation is the delineation of a novel class of moleculartargets for potential growth-modulatory drugs. The macrolide ester,rapamycin, is a potent immunosuppressant whose mechanism of action isrelated to the inhibition of cytokine-dependent T cell proliferation(Bierer et al. (1990) PNAS 87:9231-9235; Dumont et al. (1990) J Immunol144:1418-1424; Sigal et al. (1991) Transplant Proc 23:1-5; and Sigal etal. (1992) Annu Rev Immunol 110:519-560). Rapamycin specificallyinterferes with a late G₁-phase event required for the progression ofIL-2 stimulated cells into S-phase (Morice et al. (1993) J Biol Chem268:3734-3738). The location of the cell cycle arrest point induced byrapamycin hints that this drug interferes with the regulatory proteinsthat govern the G₁-to-S phase transition, particularly in lymphocytes.

As described herein, the present invention relates to the discovery ofnovel proteins of mammalian origin which are immediate downstreamtargets for FKBP/rapamycin complexes. As described below, adrug-dependent interaction trap assay was used to isolate a number ofproteins which bind the FKBP12/rapamycin complex, and which arecollectively referred to herein as “RAP-binding proteins” or “RAP-BPs”.In particular, mouse and human genes have been cloned for a protein(referred to herein as “RAPT1”) which is apparently related to the yeastTOR1 and TOR2 gene products. Furthermore, a novel ubiquitin-conjugatingenzyme (referred to herein as “rap-UBC”) has been cloned based on itsability to bind FKBP/rapamycin complexes. The present invention,therefore, makes available novel proteins (both recombinant and purifiedforms), recombinant genes, antibodies to RAP-binding proteins, and othernovel reagents and assays for diagnostic and therapeutic use. Moreover,drug discovery assays are provided for identifying agents which canmodulate the binding of one or more of the subject RAP-binding proteinswith FK506-binding proteins. Such agents can be useful therapeuticallyto alter the growth and/or differentiation of a cell, but can also beused in vitro as cell-culture additives for controlling proliferationand/or differentiation of cultured cells and tissue. Other aspects ofthe invention are described below or will be apparent to those skilledin the art in light of the present disclosure.

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single-stranded (such assense or antisense) and double-stranded polynucleotides.

The term “gene” or “recombinant gene” refers to a nucleic acidcomprising an open reading frame encoding a RAP-binding protein of thepresent invention, including both exon and (optionally) intronsequences. A “recombinant gene” refers to nucleic acid encoding aRAP-binding protein and comprising RAP-BP encoding exon sequences,though it may optionally include intron sequences which are eitherderived from a chromosomal RAP-BP gene or from an unrelated chromosomalgene. Exemplary recombinant genes encoding illustrative RAP-bindingproteins include a nucleic acid sequence represented by on of SEQ IDNos: 1, 11 or 23. The term “intron” refers to a DNA sequence present ina given RAP-BP gene which is not translated into protein and isgenerally found between exons.

As used herein, the term “transfection” refers to the introduction of anucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of the RAP-binding proteinof the present invention or where anti-sense expression occurs from thetransferred gene, the expression for a naturally-occurring form of theRAP-binding protein is disrupted.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer to circular double stranded DNA loops which, in their vector formare not bound to the chromosome. In the present specification, “plasmid”and “vector” are used interchangeably as the plasmid is the mostcommonly used form of vector. However, the invention is intended toinclude such other forms of expression vectors which serve equivalentfunctions and which become known in the art subsequently hereto.

“Transcriptional regulatory sequence” is a generic term used throughoutthe specification to refer to DNA sequences, such as initiation signals,enhancers, and promoters, which induce or control transcription ofprotein coding sequences with which they are operably linked. Inpreferred embodiments, transcription of a recombinant RAP-BP gene isunder the control of a promoter sequence (or other transcriptionalregulatory sequence) which controls the expression of the recombinantgene in a cell-type in which expression is intended. It will also beunderstood that the recombinant gene can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring form of the RAP-binding protein.

As used herein, the term “tissue-specific promoter” means a DNA sequencethat serves as a promoter, i.e., regulates expression of a selected DNAsequence operably linked to the promoter, and which effects expressionof the selected DNA sequence in specific cells of a tissue, such ascells of a lymphoid lineage, e.g. B or T lymphocytes, or alternatively,e.g. hepatic cells. In an illustrative embodiment, gene constructsutilizing lymphoid-specific promoters can be used as a part of genetherapy to provide dominant negative mutant forms of a RAP-bindingprotein to render lymphatic cells resistant to rapamycin by directingexpression of the mutant form of RAP-BP in only lymphatic tissue. Theterm also covers so-called “leaky” promoters, which regulate expressionof a selected DNA primarily in one tissue, but cause expression in othertissues as well.

As used herein, a “transgenic animal” is any animal, preferably anon-human mammal, a bird or an amphibian, in which one or more of thecells of the animal contain heterologous nucleic acid introduced by wayof human intervention, such as by trangenic techniques well known in theart. The nucleic acid is introduced into the cell, directly orindirectly by introduction into a precursor of the cell, by way ofdeliberate genetic manipulation, such as by microinjection or byinfection with a recombinant virus. The term genetic manipulation doesnot include classical cross-breeding, or in vitro fertilization, butrather is directed to the introduction of a recombinant DNA molecule.This molecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a recombinantform of a subject RAP-binding protein, e.g. either agonistic orantagonistic forms. However, transgenic animals in which the recombinantRAP-BP gene is silent are also contemplated, as for example, the FLP orCRE recombinase dependent constructs described below. The “non-humananimals” of the invention include vertebrates such as rodents, non-humanprimates, sheep, dog, cow, chickens, amphibians, reptiles, etc.Preferred non-human animals are selected from the rodent familyincluding rat and mouse, most preferably mouse, though transgenicamphibians, such as members of the Xenopus genus, and transgenicchickens can also provide important tools for understanding, forexample, embryogenesis and tissue patterning. The term “chimeric animal”is used herein to refer to animals in which the recombinant gene isfound, or in which the recombinant is expressed in some but not allcells of the animal. The term “tissue-specific chimeric animal”indicates that the recombinant RAP-BP gene is present and/or expressedin some tissues but not others.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., a RAP-binding protein), which is partly or entirelyheterologous, i.e., foreign, to the transgenic animal or cell into whichit is introduced, or, is homologous to an endogenous gene of thetransgenic animal or cell into which it is introduced, but which isdesigned to be inserted, or is inserted, into the animal's genome insuch a way as to alter the genome of the cell into which it is inserted(e.g., it is inserted at a location which differs from that of thenatural gene or its insertion results in a knockout). A transgene caninclude one or more transcriptional regulatory sequences and any othernucleic acid, such as introns, that may be necessary for optimalexpression of a selected nucleic acid.

As is well known, genes for a particular polypeptide may exist in singleor multiple copies within the genome of an individual. Such duplicategenes may be identical or may have certain modifications, includingnucleotide substitutions, additions or deletions, which all still codefor polypeptides having substantially the same activity. The term “DNAsequence encoding a RAP-binding protein” may thus refer to one or moregenes within a particular individual. Moreover, certain differences innucleotide sequences may exist between individual organisms, which arecalled alleles. Such allelic differences may or may not result indifferences in amino acid sequence of the encoded polypeptide yet stillencode a protein with the same biological activity.

“Homology” refers to sequence similarity between two peptides or betweentwo nucleic acid molecules. Homology can be determined by comparing aposition in each sequence which may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame base or amino acid, then the molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences.

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A “chimeric protein” or “fusion protein” is a fusion of a first aminoacid sequence encoding one of the subject RAP-binding proteins with asecond amino acid sequence defining a domain foreign to and notsubstantially homologous with any domain of the subject RAP-BP. Achimeric protein may present a foreign domain which is found (albeit ina different protein) in an organism which also expresses the firstprotein, or it may be an “interspecies”, “intergeneric”, etc. fusion ofprotein structures expressed by different kinds of organisms.

The term “evolutionarily related to”, with respect to nucleic acidsequences encoding RAP-binding proteins, refers to nucleic acidsequences which have arisen naturally in an organism, includingnaturally occurring mutants. Moreover, the term also refers to nucleicacid sequences which, while initially derived from naturally-occurringisoforms of RAP-binding proteins, have been altered by mutagenesis, asfor example, such combinatorial mutagenesis as described below, yetwhich still encode polypeptides that bind FKBP/rapamycin complexes, orthat retain at least one activity of the parent RAP-binding protein, orwhich are antagonists of that protein's activities.

The term “isolated” as also used herein with respect to nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs, orRNAs, respectively, that are present in the natural source of themacromolecule. For example, an isolated nucleic acid encoding one of thesubject RAP-binding proteins preferably includes no more than 10kilobases (kb) of nucleic acid sequence which naturally immediatelyflanks that particular RAP-BP gene in genomic DNA, more preferably nomore than 5 kb of such naturally occurring flanking sequences, and mostpreferably less than 1.5 kb of such naturally occurring flankingsequence. The term isolated as used herein also refers to a nucleic acidor peptide that is substantially free of cellular material, viralmaterial, or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesized.Moreover, an “isolated nucleic acid” is meant to include nucleic acidfragments which are not naturally occurring as fragments and would notbe found in the natural state.

As used herein, an “rapamycin-binding domain” refers to a polypeptidesequence which confers a binding activity for specifically interactingwith an FKBP/rapamycin complex. Exemplary rapamycin-binding domains arerepresented within the polypeptides defined by Val26-Tyr160 of SEQ IDNo. 2, Val1272-Tyr1444 of SEQ ID No. 12, Val41-Tyr173 of SEQ ID No. 14,Val1-Tyr133 of SEQ ID No. 16, and Val1-Arg133 of SEQ ID No. 18.

A “RAPT1-like polypeptide” refers to a eukaryotic cellular protein whichis a direct binding target protein for an FKBP/rapamycin complex, andwhich shares some sequence homology with a mammalian RAPT1 protein ofthe present invention. Exemplary RAPT1-like polypeptides include theyeast TOR1 and TOR2 proteins.

A “soluble protein” refers to a polypeptide which does not precipitate(e.g. at least about 95-percent, more preferably at least 99-percentremains in the supernatant) from an aqueous buffer under physiologicallyisotonic condition, for example, 0.14M NaCl or sucrose, at a proteinconcentration of as much as 10 μM, more preferably as much as 10 mM.These conditions specifically relate to the absence of detergents orother denaturants in effective concentrations.

As described below, one aspect of this invention pertains to an isolatednucleic acid comprising the nucleotide sequence encoding a RAP-bindingprotein, fragments thereof, and/or equivalents of such nucleic acids.The term nucleic acid as used herein is intended to include suchfragments and equivalents. The term equivalent is understood to includenucleotide sequences encoding functionally equivalent RAP-bindingproteins or functionally equivalent peptides which, for example, retainthe ability to bind to the FKBP/rapamycin complex, and which mayadditionally retain other activities of a RAP-binding protein such asdescribed herein. Equivalent nucleotide sequences will include sequencesthat differ by one or more nucleotide substitutions, additions ordeletions, such as allelic variants; and will also include sequencesthat differ from the nucleotide sequence of the mammalian RAPT1 genesrepresented in SEQ ID No: 1 or SEQ ID No. 11, or the nucleotide sequenceof the fungal RAPT1 protein of SEQ ID No. 13, or the nucleotide sequenceencoding the UBC enzyme represented in SEQ ID No. 23, due to thedegeneracy of the genetic code. Equivalent nucleic acids will alsoinclude nucleotide sequences that hybridize under stringent conditions(i.e., equivalent to about 20-27° C. below the melting temperature(T_(m)) of the DNA duplex formed in about 1M salt) to a nucleotidesequence of a RAPT1 protein comprising either the sequence shown in SEQID No: 2 or 12, or to a nucleotide sequence of the RAPT1 gene insert ofpIC524 (ATCC accession no. 75787). Likewise, equivalent nucleic acidsencoding homologs of the subject rap-UBC enzyme include nucleotidesequences that hybridize under stringent conditions to a nucleotidesequence represented in SEQ ID No. 23, or to a nucleotide sequence ofthe rap-UBC gene insert of SMR4-15 (ATCC accession no. 75786). In oneembodiment, equivalents will further include nucleic acid sequencesderived from, and evolutionarily related to, a nucleotide sequencecomprising that shown in either SEQ ID No. 1, or SEQ ID No. 11, or SEQID No. 13, or SEQ ID No. 23.

The amino acid sequences shown in each of SEQ ID Nos: 2 and 12 representbiologically active portions of larger full-length forms of mammalianRAPT1 proteins. In preferred embodiments, the RAPT1 polypeptide includesa binding domain for binding to FKBP/rapamycin complexes, such as therap-binding domains represented by residues 28-160 of SEQ ID No. 2, orresidues 1272-1444 of SEQ ID No. 12. In preferred embodiments, portionsof the RAPT1 protein isolated from the full-length form will retain aspecific binding affinity for an FKBP/rapamycin complex, e.g. anFKBP12/rapamycin complex, e.g. an affinity at least 50%, more preferablyat least 75%, and even more preferably at least 90% that of the bindingaffinity of a naturally-occurring form of RAPT1 for such a rapamycincomplex. A polypeptide is considered to possess a biological activity ofa RAPT1 protein if the polypeptide has one or more of the followingproperties: the ability to bind an FKBP/drug complex, e.g., anFKBP/macrolide complex, e.g., an FKBP/rapamycin complex; the ability tobind to an FKBP12/rapamycin complex; the ability to modulate assembly ofFKBP/rapamycin-complexes; the ability to regulate cell proliferation,e.g., to regulate the cell-cycle, e.g., to regulate the progression of acell through the G₁ phase. Moreover, based on sequence analysis, thebiological function of the subject RAPT1 proteins can include aphosphatidyl inositol-kinase activity, such as a PI-3-kinase activity. Aprotein also has biological activity if it is a specific agonist orantagonist of one of the above recited properties.

Likewise, the amino acid sequence shown in SEQ ID No. 24 represents abiologically active portion of a larger full-length form of a humanubiquitin-conjugating enzyme. Accordingly, preferred embodiments of thesubject rap-UBC comprise at least a portion of the amino acid sequenceof SEQ ID No. 24 (or of the rap-UBC gene insert of SMR4-15 described inExample 5) which possess either the ability to bind a FKBP/rapamycincomplex or the ability to conjugating ubiquitin to a cellular protein,or both. Given that rapamycin causes a block in the cell-cycle during G1phase, it is probable that the spectrum of biological activity of thesubject rap-UBC enzyme includes control of half-lives of certain cellcycle regulatory proteins, particularly relatively short lived proteins(e.g. proteins which have half-lives on the order of 30 minutes to 2hours). For example, the subject UBC may have the ability to mediateubiquitination of, for example, p53, myc and/or cyclins, and thereforeaffects the cellular half-life of a cell-cycle regulatory protein inproliferating cells. The binding of the rap-UBC to the FKBP/rapamycincomplex may result in sequestering of the enzyme away from its substrateproteins. Thus, rapamycin may interfere with the ubiquitin-mediateddegradation of p53 in a manner which causes cellular p53 levels to risewhich in turn inhibits progression of the G1 phase.

Moreover, it will be generally appreciated that, under certaincircumstances, it may be advantageous to provide homologs of the clonedRAP-binding proteins which function in a limited capacity as one ofeither a RAP-BP agonists or a RAP-BP antagonists, in order to eitherpromote or inhibit only a subset of the biological activities of thenaturally occurring form of the protein. Thus, specific biologicaleffects can be elicited by treatment with a homolog of limited function,and with fewer side effects relative to treatment with agonists orantagonists which are directed- to all RAP-BP related biologicalactivities. For instance, RAPT1 analogs and rap-UBC analogs can begenerated which do not bind in any substantial fashion to anFKBP/rapamycin complex, yet which retain most of the other biologicalfunctions ascribed to the naturally-occurring form of the protein. Forexample, the RAPT1 homolog might retain a kinase activity, such as aphosphatidyl inositol kinase activity, e.g. a PI-3-kinase activity.Conversely, the RAPT1 homolog may be engineered to lack a kinaseactivity, yet retain the ability to bind an FKBP/rapamycin complex. Forinstance, the FKBP/rapamycin binding portions of the RAPT1 homologs,such as the rapamycin-binding domains represented in SEQ ID Nos. 2 or12, can be used to competitively inhibit binding to rapamycin complexesby the naturally-occurring form of RAPT1. In similar fashion, rap-UBChomologs can be provided which, for example, are catalytically inactive(e.g. an active site mutant, e.g. Cys-92 to Ser) yet which still bindsan FKBP/rapamycin complex. Such a homolog is likely to actantagonistically to the role of the natural enzyme in rapamycin action

Homologs of the subject RAP-binding proteins can be generated bymutagenesis, such as by discrete point mutation(s), or by truncation.For instance, mutation can give rise to homologs which retainsubstantially the same, or merely a subset, of the biological activityof the RAP-BP from which it was derived. Alternatively, antagonisticforms of the protein can be generated which are able to inhibit thefunction of the naturally occurring form of the protein, such as bycompetitively binding to FKBP/rapamycin complexes.

The nucleotide sequence shown in SEQ ID No: 1 encodes a biologicallyactive portion of the mouse RAPT1 protein, and in particular, includes arapamycin-binding domain. Accordingly, in one embodiment of the presentinvention, the nucleic acid is a cDNA encoding a peptide including anamino acid sequence substantially homologous to that portion of theRAPT1 protein represented by SEQ ID No: 2. Preferably, the nucleic acidis a cDNA molecule comprising at least a portion of the nucleotidesequence shown in SEQ ID No: 1. Likewise, the nucleotide sequence shownin SEQ ID No. 11 encodes a biologically active portion of the humanRAPT1 protein. Thus, another embodiment of the present inventionprovides a cDNA encoding a peptide having an amino acid sequencesubstantially homologous to that portion of the RAPT1 proteinrepresented by SEQ ID No. 12. In similar fashion, the present inventionprovides a cDNA encoding at least a portion of the Candida RAPT1polypeptide of SEQ ID No. 14.

Preferred nucleic acids encode a polypeptide including an amino acidsequence which is at least 60% homologous, more preferably 70%homologous and most preferably 80% homologous with an amino acidsequence shown in one or more of SEQ ID Nos: 2, 12 or 14. Nucleic acidsencoding peptides, particularly peptides having an activity of a RAPT1protein, and comprising an amino acid sequence which is at least about90%, more preferably at least about 95%, and most preferably at leastabout 98-99% homologous with a sequence shown in either SEQ ID No: 2, 12or 14 are also within the scope of the invention, as of course areproteins which are identical to the aforementioned sequence listings. Inone embodiment, the nucleic acid is a cDNA encoding a peptide having atleast one activity of a subject RAP-binding protein. Preferably, thenucleic acid is a cDNA molecule comprising at least a portion of thenucleotide sequence represented in one of SEQ ID Nos: 2, 12 or 14. Apreferred portion of these cDNA molecules includes the coding region ofthe gene. For instance, a recombinant RAP-BP gene can include nucleotidesequences of a PCR fragment generated by amplifying the coding sequencesfor one of the RAP-BP clones of ATCC deposit No: 75787.

The nucleotide sequence shown in SEQ ID No: 23 encodes a biologicallyactive portion of the human rap-UBC enzyme. Accordingly, in oneembodiment of the present invention, the nucleic acid is a cDNA encodinga peptide including an amino acid sequence substantially homologous tothat portion of the rap-UBC protein represented by SEQ ID No: 24.Preferably, the nucleic acid is a cDNA molecule comprising at least aportion of the nucleotide sequence shown in SEQ ID No: 23. Preferrednucleic acids encode a peptide comprising an amino acid sequence whichis at least 60% homologous, more preferably 70% homologous and mostpreferably 80% homologous with an amino acid sequence shown in SEQ IDNo: 24. Nucleic acids encoding polypeptides, particularly those having aubiquitin conjugating activity, and comprising an amino acid sequencewhich is at least about 90%, more preferably at least about 95%, andmost preferably at least about 98-99% homologous with a sequence shownin SEQ ID No: 24 are also within the scope of the invention.

In a further embodiment of the invention, the recombinant RAP-BP genescan further include, in addition to the amino acid sequence shown in SEQID No. 2, 12 or 24, additional nucleotide sequences which encode aminoacids at the C-terminus and N-terminus of the protein though not shownin those sequence listings. For instance, the recombinant RAPT1 gene caninclude nucleotide sequences of a PCR fragment generated by amplifyingthe RAPT1 coding sequence of pIC524 using sets of primers such describedin Example 4. Additionally, in light of the present disclosure, it willbe possible using no more than routine experimentation to isolate from,for example, a cDNA library, the remaining 5′ sequences of RAPT1, suchas by RACE PCR using primers designed from the present sequences. Inparticular, the invention contemplates a recombinant RAPT1 gene encodingthe full-length RAPT1 protein. Yet another embodiment of the inventionincludes nucleic acids that encode isoforms of the mouse or human RAPT1,especially isoforms (e.g. splicing variants, allelic variants, etc.)that are capable of binding with the FKBP12/rapamycin complex. Suchisoforms, as well as other members of the larger family of RAP-bindingproteins, can be isolated using the drug-dependent interaction trapassays described in further detail below.

Another aspect of the invention provides a nucleic acid that hybridizesunder high or low stringency conditions to a nucleic acid which encodesa peptide having at least a portion of an amino acid sequencerepresented by one of SEQ ID Nos.: 2, 12, 14 or 24. Appropriatestringency conditions which promote DNA hybridization, for example, 6.0×sodium chloride/sodium citrate (SSC) at about 45° C., followed by a washof 2.0×SSC at 50° C., are known to those skilled in the art or can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. For example, the salt concentration in the washstep can be selected from a low stringency of about 2.0×SSC at 50° C. toa high stringency of about 0.2×SSC at 50° C. In addition, thetemperature in the wash step can be increased from low stringencyconditions at room temperature, about 22° C., to high stringencyconditions at about 65° C.

Nucleic acids having a sequence which differs from the nucleotidesequence shown in any of SEQ ID Nos: 1, 11, 13 or 23 due to degeneracyin the genetic code are also within the scope of the invention. Suchnucleic acids encode functionally equivalent peptides (i.e., a peptidehaving a biological activity of a RAP-binding protein) but that differin sequence from the appended sequence listings due to degeneracy in thegenetic code. For example, a number of amino acids are designated bymore than one triplet. Codons that specify the same amino acid, orsynonyms (for example, CAU and CAC each encode histidine) may result in“silent” mutations which do not affect the amino acid sequence of theRAP-binding protein. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequences of thesubject RAP-binding proteins will exist among vertebrates. One skilledin the art will appreciate that these variations in one or morenucleotides (up to about 3-5% of the nucleotides) of the nucleic acidsencoding polypeptides having an activity of a RAP-binding protein mayexist among individuals of a given species due to natural allelicvariation. Any and all such nucleotide variations and resulting aminoacid polymorphisms are within the scope of this invention.

The present invention also provides nucleic acid encoding only a portionof a RAPT1 protein, such as the rapamycin-binding domain. As usedherein, a fragment of a nucleic acid encoding such a portion of aRAP-binding protein refers to a nucleotide sequence having fewernucleotides than the nucleotide sequence encoding the entire amino acidsequence of a full-length RAP-binding protein, yet which still includesenough of the coding sequence so as to encode a polypeptide which iscapable of binding to an FKBP/rapamycin complex. Moreover, nucleic acidfragments within the scope of the invention include those fragmentscapable of hybridizing under high or low stringency conditions withnucleic acids from other vertebrate species, particularly other mammals,and can be used in screening protocols to detect homologs, of thesubject RAP-binding proteins. Nucleic acids within the scope of theinvention may also contain linker sequences, modified restrictionendonuclease sites and other sequences useful for molecular cloning,expression or purification of recombinant peptides derived fromRAP-binding proteins.

As indicated by the examples set out below, a nucleic acid encoding aRAP-binding protein may be obtained from mRNA present in any of a numberof cells from a vertebrate organism, particularly from mammals, e.g.mouse or human. It should also be possible to obtain nucleic acidsencoding RAP-binding proteins from genomic DNA obtained from both adultsand embryos. For example, a gene encoding a RAP-binding protein can becloned from either a cDNA or a genomic library in accordance withprotocols herein described, as well as those generally known in the art.For instance, a cDNA encoding a RAPT1 protein, particularly otherisoforms of the RAPT1 proteins represented by either SEQ ID No. 2 or 12,can be obtained by isolating total mRNA from a mammalian cell, e.g. ahuman cell, generating double stranded cDNAs from the total mRNA,cloning the cDNA into a suitable plasmid or bacteriophage vector, andisolating RAPT1 clones using any one of a number of known techniques,e.g. oligonucleotide probes or western blot analysis. Genes encodingproteins related to the subject RAP-binding proteins can also be clonedusing established polymerase chain reaction techniques in accordancewith the nucleotide sequence information provided by the invention. Thenucleic acid of the invention can be DNA or RNA.

Another aspect of the invention relates to the use of the isolatednucleic acid in “antisense” therapy. As used herein, “antisense” therapyrefers to administration or in situ generation of oligonucleotide probesor their derivatives which specifically hybridizes (e.g. binds) undercellular conditions, with the cellular mRNA and/or genomic DNA encodinga RAP-binding protein so as to inhibit expression of that protein, asfor example by inhibiting transcription and/or translation. The bindingmay be by conventional base pair complementarity, or, for example, inthe case of binding to DNA duplexes, through specific interactions inthe major groove of the double helix. In general, “antisense” therapyrefers to the range of techniques generally employed in the art, andincludes any therapy which relies on specific binding to oligonucleotidesequences.

An antisense construct of the present invention can be delivered, forexample, as an expression plasmid which, when transcribed in the cell,produces RNA which is complementary to at least a unique portion of thecellular mRNA which encodes a RAP-binding protein. Alternatively, theantisense construct can be an oligonucleotide probe which is generatedex vivo and which, when introduced into the cell causes inhibition ofexpression by hybridizing with the mRNA and/or genomic sequences of aRAP-BP gene. Such oligonucleotide probes are preferably modifiedoligonucleotides which are resistant to endogenous nucleases, e.g.exonucleases and/or endonucleases, and is therefore stable in vivo.Exemplary nucleic acid molecules for use as antisense oligonucleotidesare phosphoramidate, phosphothioate and methylphosphonate analogs of DNA(see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775).Additionally, general approaches to constructing oligomers useful inantisense therapy have been reviewed, for example, by van der Krol etal. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res48:2659-2668.

Accordingly, the modified oligomers of the invention are useful intherapeutic, diagnostic, and research contexts. In therapeuticapplications, the oligomers are utilized in a manner appropriate forantisense therapy in general. For such therapy, the oligomers of theinvention can be formulated for a variety of loads of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remmington's PharmaceuticalSciences, Meade Publishing Co., Easton, Pa. For systemic administration,injection is preferred, including intramuscular, intravenous,intraperitoneal, and subcutaneous for injection, the oligomers of theinvention can be formulated in liquid solutions, preferably inphysiologically compatible buffers such as Hank's solution or Ringer'ssolution. In addition, the oligomers may be formulated in solid form andredissolved or suspended immediately prior to use. Lyophilized forms arealso included.

Systemic administration can also be by transmucosal or transdermalmeans, or the compounds can be administered orally. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art, and include, for example, for transmucosaladministration bile salts and fusidic acid derivatives. In addition,detergents may be used to facilitate permeation. Transmucosaladministration may be through nasal sprays or using suppositories. Fororal administration, the oligomers are formulated into conventional oraladministration forms such as capsules, tablets, and tonics. For topicaladministration, the oligomers of the invention are formulated intoointments, salves, gels, or creams as generally known in the art.

In addition to use in therapy, the oligomers of the invention may beused as diagnostic reagents to detect the presence or absence of thetarget DNA or RNA sequences to which they specifically bind. Suchdiagnostic tests are described in further detail below.

Likewise, the antisense constructs of the present invention, byantagonizing the normal biological activity of a RAP-binding protein,can be used in the manipulation of tissue, e.g. tissue proliferationand/or differentiation, both for in vivo and ex vivo tissue culturesystems.

This invention also provides expression vectors containing a nucleicacid encoding a RAP-binding protein of the present invention, operablylinked to at least one transcriptional regulatory sequence. Operablylinked is intended to mean that the nucleotide sequence is linked to aregulatory sequence in a manner which allows expression of thenucleotide sequence. Regulatory sequences are art-recognized and areselected to direct expression of a recombinant RAP-binding protein.Accordingly, the term transcriptional regulatory sequence includespromoters, enhancers and other expression control elements. Suchregulatory sequences are described in Goeddel; Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). For instance, any of a wide variety of expression controlsequences-sequences that control the expression of a DNA sequence whenoperatively linked to it may be used in these vectors to express DNAsequences encoding the RAP-binding proteins of this invention. Suchuseful expression control sequences, include, for example, the early andlate promoters of SV40, adenovirus or cytomegalovirus immediate earlypromoter, the lac system, the trp system, the TAC or TRC system, T7promoter whose expression is directed by T7 RNA polymerase, the majoroperator and promoter regions of phage lambda, the control regions forfd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, thepromoters of the yeast α-mating factors, the polyhedron promoter of thebaculovirus system and other sequences known to control the expressionof genes of prokaryotic or eukaryotic cells or their viruses, andvarious combinations thereof. It should be understood that the design ofthe expression vector may depend on such factors as the choice of thehost cell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other proteins encoded by thevector, such as antibiotic markers, should also be considered. In oneembodiment, the expression vector includes a recombinant gene encoding apolypeptide which mimics or otherwise agonizes the action of aRAP-binding protein, or alternatively, which encodes a polypeptide thatantagonizes the action of an authentic RAP-binding protein. Suchexpression vectors can be used to transfect cells and thereby producepolypeptides, including fusion proteins, encoded by nucleic acids asdescribed herein.

Moreover, the gene constructs of the present invention can also be usedas a part of a gene therapy protocol to deliver nucleic acids encodingeither an agonistic or antagonistic form of one or more of the subjectRAP-binding proteins. Thus, another aspect of the invention featuresexpression vectors for in vivo transfection and expression of aRAP-binding protein in particular cell types so as to reconstitute thefunction of, or alternatively, abrogate the function of one or more ofthe subject RAP-binding proteins in a cell in which that protein orother transcriptional regulatory proteins to which it bind aremisexpressed. For example, gene therapy can be used to deliver a geneencoding a rapamycin-insensitive RAP-binding protein in order to rendera particular tissue or cell-type resistant to rapamycin inducedcell-cycle arrest.

Expression constructs of the subject RAP-binding proteins, and mutantsthereof, may be administered in any biologically effective carrier, e.g.any formulation or composition capable of effectively delivering theRAP-BP gene to cells in vivo. Approaches include insertion of thesubject gene in viral vectors including recombinant retroviruses,adenovirus, adeno-associated virus, and herpes simplex virus-1, orrecombinant bacterial or eukaryotic plasmids. Viral vectors transfectcells directly; plasmid DNA can be delivered with the help of, forexample, cationic liposomes (lipofectin) or derivatized (e.g. antibodyconjugated), polylysine conjugates, gramacidin S, artificial viralenvelopes or other such intracellular carriers, as well as directinjection of the gene construct or CaPO₄ precipitation carried out invivo. It will be appreciated that because transduction of appropriatetarget cells represents the critical first step in gene therapy, choiceof the particular gene delivery system will depend on such factors asthe phenotype of the intended target and the route of administration,e.g. locally or systemically. Furthermore, it will be recognized thatthe particular gene construct provided for in vivo transduction ofRAP-BP expression are also useful for in vitro transduction of cells,such as in diagnostic assays.

A preferred approach for in vivo introduction of nucleic acid into acell is by use of a viral vector containing nucleic acid, e.g. a cDNA,encoding the particular form of the RAP-binding protein desired.Infection of cells with a viral vector has the advantage that a largeproportion of the targeted cells can receive the nucleic acid.Additionally, molecules encoded within the viral vector, e.g., by a cDNAcontained in the viral vector, are expressed efficiently in cells whichhave taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery system of choice for thetransfer of exogenous genes in vivo, particularly into humans. Thesevectors provide efficient delivery of genes into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. A major prerequisite for the use of retroviruses is toensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population. Thedevelopment of specialized cell lines (termed “packaging cells”) whichproduce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arewell characterized for use in gene transfer for gene therapy purposes(for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol, env) has been replaced by nucleic acid encoding oneof the subject receptors rendering the retrovirus replication defective.

The replication defective retrovirus is then packaged into virions whichcan be used to infect a target cell through the use of a helper virus bystandard techniques. Protocols for producing recombinant retrovirusesand for infecting cells in vitro or in vivo with such viruses can befound in Current Protocols in Molecular Biology, Ausubel, F. M. et al.(eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 andother standard laboratory manuals. Examples of suitable retrovirusesinclude pLJ, pZIP, pWE and pEM which are well known to those skilled inthe art. Examples of suitable packaging virus lines for preparing bothecotropic and amphotropic retroviral systems include ψCrip, ψCre, ψ2 andψAm. Retroviruses have been used to introduce a variety of genes intomany different cell types, including lymphocytes, in vitro and/or invivo (see for example Eglitis, et al. (1985) Science 230:1395-1398;Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464;Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentanoet al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al.(1991) Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991)Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al. (1991) Science254:1802-1805; van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA89:7640-7644; Kay et al. (1992) Human Gene Therapy 3:641-647; Dai et al.(1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J.Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No.4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCTApplication WO 89/05345; and PCT Application WO 92/07573).

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234 andWO94/06920). For instance, strategies for the modification of theinfection spectrum of retroviral vectors include: coupling antibodiesspecific for cell surface antigens to the viral env protein (Roux et al.(1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255;and Goud et al. (1983) Virology 163:251-254); or coupling cell surfacereceptor ligands to the viral env proteins (Neda et al. (1991) J BiolChem 266:14143-14146). Coupling can be in the form of the chemicalcross-linking with a protein or other variety (e.g. lactose to convertthe env protein to an asialoglycoprotein), as well as by generatingfusion proteins (e.g. single-chain antibody/env fusion proteins). Thistechnique, while useful to limit or otherwise direct the infection tocertain tissue types, can also be used to convert an ecotropic vector into an amphotropic vector.

Moreover, use of retroviral gene delivery can be further enhanced by theuse of tissue- or cell-specific transcriptional regulatory sequenceswhich control expression of the RAP-BP gene of the retroviral vector.

Another viral gene delivery system useful in the present inventionutilizes adenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes and expresses a gene product ofinterest but is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. See for example Berkner et al. (1988)BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; andRosenfeld et al. (1992) Cell 68:143-155. Suitable adenoviral vectorsderived from the adenovirus strain Ad type 5 dl324 or other strains ofadenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled inthe art. Recombinant adenoviruses can be advantageous in certaincircumstances in that they are not capable of infecting nondividingcells and can be used to infect a wide variety of cell types.Furthermore, the virus particle is relatively stable and amenable topurification and concentration, and as above, can be modified so as toaffect the spectrum of infectivity. Additionally, introduced adenoviralDNA (and foreign DNA contained therein) is not integrated into thegenome of a host cell but remains episomal, thereby avoiding potentialproblems that can occur as a result of insertional mutagenesis insituations where introduced DNA becomes integrated into the host genome(e.g., retroviral DNA). Moreover, the carrying capacity of theadenoviral genome for foreign DNA is large (up to 8 kilobases) relativeto other gene delivery vectors (Berkner et al. cited supra; Haj-Ahmandand Graham (1986) J. Virol. 57:267). Most replication-defectiveadenoviral vectors currently in use and therefore favored by the presentinvention are deleted for all or parts of the viral E1 and E3 genes butretain as much as 80% of the adenoviral genetic material (see, e.g.,Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham etal. in Methods in Molecular Biology, E. J. Murray, Ed. (Humana, Clifton,N.J., 1991) vol. 7. pp. 109-127). Expression of the inserted RAP-BP genecan be under control of, for example, the E1A promoter, the major latepromoter (MLP) and associated leader sequences, the E3 promoter, orexogenously added promoter sequences.

Yet another viral vector system useful for delivery of the subjectRAP-BP gene is the adeno-associated virus (AAV). Adeno-associated virusis a naturally occurring defective virus that requires another virus,such as an adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.(1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al.(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of anRAP-binding protein in the tissue of an animal. Most nonviral methods ofgene transfer rely on normal mechanisms used by mammalian cells for theuptake and intracellular transport of macromolecules. In preferredembodiments, non-viral gene delivery systems of the present inventionrely on endocytic pathways for the uptake of the subject RAP-BP gene bythe targeted cell. Exemplary gene delivery systems of this type includeliposomal derived systems, poly-lysine conjugates, and artificial viralenvelopes.

In a representative embodiment, a gene encoding one of the subjectRAP-binding proteins can be entrapped in liposomes bearing positivecharges on their surface (e.g., lipofectins) and (optionally) which aretagged with antibodies against cell surface antigens of the targettissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551; PCT publicationWO91/06309; Japanese patent application 1047381; and European patentpublication EP-A-43075). For example, lipofection of cells can becarried out using liposomes tagged with monoclonal antibodies againstany cell surface antigen present on, for example, T-cells.

In clinical settings, the gene delivery systems for the therapeuticRAP-BP gene can be introduced into a patient by any of a number ofmethods, each of which is familiar in the art. For instance, apharmaceutical preparation of the gene delivery system can be introducedsystemically, e.g. by intravenous injection, and specific transductionof the protein in the target cells occurs predominantly from specificityof transfection provided by the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.In other embodiments, initial delivery of the recombinant gene is morelimited with introduction into the animal being quite localized. Forexample, the gene delivery vehicle can be introduced by catheter (seeU.S. Pat. No. 5,328,470) or by stereotactic injection (e.g. Chen et al.(1994) PNAS 91: 3054-3057).

The pharmaceutical preparation of the gene therapy construct can consistessentially of the gene delivery system in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery system can beproduced intact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

Another aspect of the present invention concerns recombinant RAP-bindingproteins which are encoded by genes derived from eukaryotic cells, e.g.mammalian cells, e.g. cells from humans, mice, rats, rabbits, or pigs.The term “recombinant protein” refers to a protein of the presentinvention which is produced by recombinant DNA techniques, whereingenerally DNA encoding, for example, the RAPT1 protein, is inserted intoa suitable expression vector which is in turn used to transform a hostcell to produce the heterologous protein. Moreover, the phrase “derivedfrom”, with respect to a recombinant gene encoding the recombinantRAP-binding protein, is meant to include within the meaning of“recombinant protein” those proteins having an amino acid sequence of anative RAP-binding protein, or an amino acid sequence similar thereto,which is generated by mutation so as to include substitutions and/ordeletions relative to a naturally occurring form of the RAP-bindingprotein of a organism. Recombinant RAPT1 proteins preferred by thepresent invention, in addition to those having an amino acid sequence ofa native RAPT1 protein, comprise amino acid sequences which are at least70% homologous, more preferably 80% homologous and most preferably 90%homologous with an amino acid sequence shown in one of SEQ ID No: 2, 12or 14. A polypeptide having a biological activity of a RAPT1 protein andwhich comprises an amino acid sequence that is at least about 95%, morepreferably at least about 98%, and most preferably are identical to asequence represented in one of SEQ ID No: 2, 12 or 14 are also withinthe scope of the invention.

Likewise, preferred embodiments of recombinant rap-UBC proteins includean amino acid sequence which is at least 70% homologous, more preferably80% homologous, and most preferably 90% homologous with an amino acidsequence represented by SEQ ID No. 24. Recombinant rap-UBC proteinswhich are identical, or substantially identical (e.g. 95 to 98%homologous) with an amino acid sequence of SEQ ID No. 24 are alsospecifically contemplated by the present invention.

In addition, the invention expressly encompasses recombinant RAPT1proteins produced from the ATCC deposited clones described in Example 4,e.g. from ATCC deposit number 75787, as well as recombinantubiquitin-conjugating enzymes produced from ATCC deposit number 75786,described in Example 5.

The present invention further pertains to recombinant forms of thesubject RAP-binding proteins which are evolutionarily related to aRAP-binding protein represented in one of SEQ ID No: 2, 12 or 24, thatis, not identical, yet which are capable of functioning as an agonist oran antagonist of at least one biological activity of a RAP-bindingprotein. The term “evolutionarily related to”, with respect to aminoacid sequences of recombinant RAP-binding proteins, refers to proteinswhich have amino acid sequences that have arisen naturally, as well asto mutational variants which are derived, for example, by recombinantmutagenesis.

Another aspect of the present invention pertains to methods of producingthe subject RAP-binding proteins. For example, a host cell transfectedwith a nucleic acid vector directing expression of a nucleotide sequenceencoding the subject RAPT1 protein or rap-UBC can be cultured underappropriate conditions to allow expression of the peptide to occur. Thepeptide may be secreted and isolated from a mixture of cells and mediumcontaining the recombinant protein. Alternatively, the peptide may beretained cytoplasmically, as the naturally occurring forms of thesubject RAP-binding proteins are believed to be, and the cellsharvested, lysed and the protein isolated. A cell culture includes hostcells, media and other byproducts. Suitable media for cell culture arewell known in the art. The recombinant RAP-binding proteins can beisolated from cell culture medium, host cells, or both using techniquesknown in the art for purifying proteins including ion-exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis, and immunoaffinity purification with antibodiesspecific for a RAP-binding protein. In one embodiment, the RAP-bindingprotein is a fusion protein containing a domain which facilitates itspurification, such as a RAPT1-GST fusion protein or a rapUBC-GST fusionprotein.

The present invention also provides host cells transfected with a RAP-BPgene for expressing a recombinant form of a RAP-binding protein. Thehost cell may be any prokaryotic or eukaryotic cell. Thus, a nucleotidesequence derived from the cloning of the RAP-binding proteins of thepresent invention, encoding all or a selected portion of a protein, canbe used to produce a recombinant form of a RAP-BP via microbial oreukaryotic cellular processes. Ligating a polynucleotide sequence into agene construct, such as an expression vector, and transforming ortransfecting host cells with the vector are standard procedures used inproducing other well-known proteins, e.g. insulin, interferons, p53,myc, cyclins and the like. Similar procedures, or modifications thereof,can be employed to prepare recombinant RAP-binding proteins, or portionsthereof, by microbial means or tissue-culture technology in accord withthe subject invention. Host cells suitable for expression of arecombinant RAP-binding protein can be selected, for example, fromamongst eukaryotic (yeast, avian, insect or mammalian) or prokaryotic(bacterial) cells.

The recombinant RAP-BP gene can be produced by ligating nucleic acidencoding a RAP-binding protein, or a portion thereof, into a vectorsuitable for expression in either prokaryotic cells, eukaryotic cells,or both. Expression vectors for production of recombinant forms ofRAP-binding proteins include plasmids and other vectors. For instance,suitable vectors for the expression of a RAP-BP include plasmids of thetypes: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derivedplasmids, pBTac-derived plasmids and pUC-derived plasmids for expressionin prokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIPS, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al. (1983) inExperimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused.

Preferred mammalian expression vectors contain prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription regulatory sequences that cause expression of arecombinant RAP-BP gene in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo,pRc/CMV, pSV2 gpt, pSV2 neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7,pko-neo and pHyg derived vectors are examples of mammalian expressionvectors suitable for transfection of eukaryotic cells. Some of thesevectors are modified with sequences from bacterial plasmids, such aspBR322, to facilitate replication and drug resistance selection in bothprokaryotic and eukaryotic cells. Alternatively, derivatives of virusessuch as the bovine papilloma virus (BPV-1), or Epstein-Barr virus(pHEBo, pREP-derived and p205) can be used for transient expression ofproteins in eukaryotic cells. Examples of other viral (includingretroviral) expression systems can be found above in the description ofgene therapy delivery systems.

In some instances, it may be desirable to express a recombinantRAP-binding protein by the use of a baculovirus expression system (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derivedvectors (such as the β-gal containing pBlueBac III).

The various methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art. For othersuitable expression systems for both prokaryotic and eukaryotic cells,as well as general recombinant procedures, see Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press: 1989) Chapters 16 and 17.

When expression of a portion of one of the subject RAP-binding proteinsis desired, i.e. a trunction mutant, such as the RAPT1 polypeptides ofSEQ ID Nos.2, 12 or 14, it may be necessary to add a start codon (ATG)to the oligonucleotide fragment containing the desired sequence to beexpressed. It is well known in the art that a methionine at theN-terminal position can be enzymatically cleaved by the use of theenzyme methionine aminopeptidase (MAP). MAP has been cloned from E. coli(Ben-Bassat et al. (1987) J. Bacteriol. 169:751-757) and Salmonellatyphimurium and its in vitro activity has been demonstrated onrecombinant proteins (Miller et al. (1987) PNAS 84:2718-1722).Therefore, removal of an N-terminal methionine, if desired, can beachieved either in vivo by expressing RAP-BP-derived polypeptides in ahost which produces MAP (e.g., E. coli or CM89 or S. cerevisiae), or invitro by use of purified MAP (e.g., procedure of Miller et al., supra).

Alternatively, the coding sequences for the polypeptide can beincorporated as a part of a fusion gene so as to be covalently linkedin-frame with a second nucleotide sequence encoding a differentpolypeptide. This type of expression system can be useful, for instance,where it is desirable to produce an immunogenic fragment of aRAP-binding protein. For example, the VP6 capsid protein of rotaviruscan be used as an immunologic carrier protein for portions of the RAPT1polypeptide, either in the monomeric form or in the form of a viralparticle. The nucleic acid sequences corresponding to the portion of theRAPT1 protein to which antibodies are to be raised can be incorporatedinto a fusion gene construct which includes coding sequences for a latevaccinia virus structural protein to produce a set, of recombinantviruses expressing fusion proteins comprising a portion of the proteinRAPT1 as part of the virion. It has been demonstrated with the use ofimmunogenic fusion proteins utilizing the Hepatitis B surface antigenfusion proteins that recombinant Hepatitis B virions can be utilized inthis role as well. Similarly, chimeric constructs coding for fusionproteins containing a portion of an RAPT1 protein and the polioviruscapsid protein can be created to enhance immunogenicity of the set ofpolypeptide antigens (see, for example, EP Publication No. 0259149; andEvans et al. (1989) Nature 339:385; Huang et al. (1988) J. Virol.62:3855; and Schlienger et al. (1992). J. Virol. 66:2). The subjectubiquitin-conjugating enzyme can be manipulated as an immunogen in likefashion.

The Multiple Antigen Peptide system for peptide-based immunization canalso be utilized, wherein a desired portion of a RAP-binding protein isobtained directly from organo-chemical synthesis of the peptide onto anoligomeric branching lysine core (see, for example, Posnett et al.(1988) JBC 263:1719 and Nardelli et al. (1992) J. Immunol. 148:914).Antigenic determinants of the RAP-binding proteins can also be expressedand presented by bacterial cells.

In addition to utilizing fusion proteins to enhance immunogenicity, itis widely appreciated that fusion proteins can also facilitate theexpression and purification of proteins, such as any one of theRAP-binding proteins of the present invention. For example, aRAP-binding protein can be generated as a glutathione-S-transferase(GST) fusion protein. Such GST fusion proteins can simplify purificationof a RAP-binding protein, as for example by affinity purification usingglutathione-derivatized matrices (see, for example, Current Protocols inMolecular Biology, eds. Ausabel et al. (N.Y.: John Wiley & Sons, 1991)).In another embodiment, a fusion gene coding for a purification leadersequence, such as a peptide leader sequence comprising apoly-(His)/enterokinase cleavage sequence, can be added to theN-terminus of the desired portion of a RAP-binding protein in order topermit purification of the poly(His)-fusion protein by affinitychromatography using a Ni²⁺ metal resin. The purification leadersequence can then be subsequently removed by treatment with enterokinase(e.g., see Hochuli et al. (1987) J. Chromatography 411:177; andJanknecht et al. PNAS 88:8972).

Techniques for making fusion genes are known to those skilled in theart. Essentially, the joining of various DNA fragments coding fordifferent polypeptide sequences is performed in accordance withconventional techniques, employing blunt-ended or stagger-ended terminifor ligation, restriction enzyme digestion to provide for appropriatetermini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which are subsequently annealed togenerate a chimeric gene sequence (see, for example, Current Protocolsin Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

The present invention also makes available purified, or otherwiseisolated forms of the subject RAP-binding proteins which is isolatedfrom, or otherwise substantially free of other cellular proteins,especially FKBP or other rapamycin binding proteins, as well asubiquitin and ubiquitin-dependent enzymes, signal transduction, andcell-cycle regulatory proteins, which may be normally associated withthe RAP-binding protein. The term “substantially free of other cellularor viral proteins” (also referred to herein as “contaminating proteins”)or “substantially pure or purified preparations” are defined asencompassing preparations of RAP-binding proteins having less than 20%(by dry weight) contaminating protein, and preferably having less than5% contaminating protein. Functional forms of the subject RAP-bindingproteins can be prepared, for the first time, as purified preparationsby using recombinant proteins as described herein. Alternatively, thesubject RAP-binding proteins can be isolated by affinity purificationusing, for example, matrix bound FKBP/rapamycin protein. By “purified”,it is meant, when referring to a peptide or DNA or RNA sequence, thatthe indicated molecule is present in the substantial absence of otherbiological macromolecules, such as other proteins (particularly FK506binding proteins, as well as other contaminating proteins). The term“purified” as used herein preferably means at least 80% by dry weight,more preferably in the range of 95-99% by weight, and most preferably atleast 99% by weight, of biological macromolecules of the same typepresent (but water, buffers, and other small molecules, especiallymolecules having a molecular weight of less than 5000, can be present).The term “pure” as used herein preferably has the same numerical limitsas “purified” immediately above. “Isolated” and “purified” do notencompass either natural materials in their native state or naturalmaterials that have been separated into components (e.g., in anacrylamide gel) but not obtained either as pure (e.g. lackingcontaminating proteins, or chromatography reagents such as denaturingagents and polymers, e.g. acrylamide or agarose) substances orsolutions.

Furthermore, isolated peptidyl portions of the subject RAP-bindingproteins can also be obtained by screening peptides recombinantlyproduced from the corresponding fragment of the nucleic acid encodingsuch peptides. In addition, fragments can be chemically synthesizedusing techniques known in the art such as conventional Merrifield solidphase f-Moc or t-Boc chemistry. For example, a RAP-binding protein ofthe present invention may be arbitrarily divided into fragments ofdesired length with no overlap of the fragments, or preferably dividedinto overlapping fragments of a desired length. The fragments can beproduced (recombinantly or by chemical synthesis) and tested to identifythose peptidyl fragments which can function as either agonists orantagonists of a RAP-binding protein activity, such as by microinjectionassays or in vitro protein binding assays. In an illustrativeembodiment, peptidyl portions of a RAP-binding protein, such as RAPT1 orrapUBC, can be tested for FKBP/rapamycin-binding activity.

It will also be possible to modify the structure of a RAP-bindingprotein for such purposes as enhancing therapeutic or prophylacticefficacy, or stability (e.g., ex vivo shelf life and resistance toproteolytic degradation in vivo). Such modified peptides, when designedto retain at least one activity of the naturally-occurring form of theprotein, are considered functional equivalents of the RAP-bindingprotein described in more detail herein. Such modified peptide can beproduced, for instance, by amino acid substitution, deletion, oraddition.

For example, it is reasonable to expect that an isolated replacement ofa leucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid (i.e. conservative mutations) will nothave a major effect on the folding of the protein, and may or may nothave much of an effect on the biological activity of the resultingmolecule. Conservative replacements are those that take place within afamily of amino acids that are related in their side chains. Geneticallyencoded amino acids are can be divided into four families: (1)acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3)nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar=glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. In similar fashion, the amino acid repertoire can begrouped as (1) acidic=aspartate, glutamate; (2) basic=lysine, argininehistidine, (3) aliphatic=glycine, alanine, valine, leucine, isoleucine,serine, threonine, with serine and threonine optionally be groupedseparately as aliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine,tryptophan; (5) amide=asparagine, glutamine; and (6)sulfur-containing=cysteine and methionine (see, for example,Biochemistry, 2nd ed., Ed. by L. Stryer, W H Freeman and Co.: 1981).Alternatively, amino acid replacement can be based on steric criteria,e.g. isosteric replacements, without regard for polarity or charge ofamino acid sidechains. Whether a change in the amino acid sequence of apeptide results in a functional RAP-BP homolog (e.g. functional in thesense that it acts to mimic or antagonize the wild-type form) can bereadily determined by assessing the ability of the variant peptide toproduce a response in cells in a fashion similar to the wild-type RAP-BPor competitively inhibit such a response. Peptides in which more thanone replacement has taken place can readily be tested in the samemanner.

This invention further contemplates a method of generating sets ofcombinatorial mutants of RAP-binding proteins, e.g. of RAPT1 proteinsand/or rap-UBC enzymes, as well as truncation mutants, thereof and isespecially useful for identifying variant sequences (e.g RAP-BPhomologs) that are functional in regulating rapamycin-mediated effects,as well as other aspects of cell growth or differentiation. In similarfashion, RAP-BP homologs can be generated by the present combinatorialapproach which are antagonists in that they are able to interfere withthe normal cellular functions of authentic forms of the protein.

One purpose for screening such combinatorial libraries is, for example,to isolate novel RAP-BP homologs from the library which function in thecapacity as one of either an agonists or an antagonist of the biologicalactivities of the wild-type (“authentic”) protein, or alternatively,which possess novel biological activities all together. To illustrate,RAPT1 homologs can be engineered by the present method to providehomologs which are unable to bind to the FKBP/rapamycin complex, yetstill retain at least a portion of the normal cellular activityassociated with authentic RAPT1. Thus, combinatorially-derived homologscan be generated to provide rapamycin-resistance. Such proteins, whenexpressed from recombinant DNA constructs, can be used in gene therapyprotocols.

Likewise, mutagenesis can give rise to RAP-BP homologs which haveintracellular half-lives dramatically different than the correspondingwild-type protein. For example, the altered protein can be renderedeither more stable or less stable to proteolytic degradation or othercellular process which result in destruction of, or otherwiseinactivation of, the authentic RAP-binding protein. Such homologs, andthe genes which encode them, can be utilized to alter the envelope ofexpression of a particular RAP-BP by modulating the half-life of theprotein. For instance, a short half-life can give rise to more transientRAPT1 biological effects and, when part of an inducible expressionsystem, can allow tighter control of recombinant RAPT1 levels within thecell. As above, such proteins, and particularly their recombinantnucleic acid constructs, can be used in gene therapy protocols.

In an illustrative embodiment of this method, the amino acid sequencesfor a population of RAP-BP homologs, or other related proteins, arealigned, preferably to promote the highest homology possible. Such apopulation of variants can include, for example, RAPT1 homologs from oneor more species, e.g. a sequence alignment of the mouse and human RAPT1proteins represented by SEQ ID Nos. 2 and 12, or different RAP-BPisoforms from the same species, e.g. different human RAPT1 isoforms.Amino acids which appear at each position of the sequence alignment canbe selected to create a degenerate set of combinatorial sequences.

In a preferred embodiment, the combinatorial RAP-BP library is producedby way of a degenerate library of genes encoding a library ofpolypeptides which each include at least a portion of potential RAP-BPsequences, e.g. the portion of RAPT1 represented by SEQ ID No:2 or 12,or the portion of rap-UBC represented by SEQ ID No. 24. A mixture ofsynthetic oligonucleotides can be enzymatically ligated into genesequences such that the degenerate set of potential RAP-BP sequences areexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g. for phage display) containing the RAP-BPsequence library therein.

There are many ways by which the library of RAP-BP homologs can begenerated from a degenerate oligonucleotide sequence. For instance,chemical synthesis of a degenerate gene sequence can be carried out inan automated DNA synthesizer, and the synthetic genes then ligated intoan appropriate gene for expression. The purpose of a degenerate set ofRAP-BP genes is to provide, in one mixture, all of the sequencesencoding the desired set of potential RAP-BP sequences. The synthesis ofdegenerate oligonucleotides is well known in the art (see, for example,Narang, S A (1983) Tetrahedron 39:3; Itakura et al. (1981) RecombinantDNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. A G Walton,Amsterdam: Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev.Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.(1983) Nucleic Acid Res. 11:477. Such techniques have been employed inthe directed evolution of other proteins (see, for example, Scott et al.(1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433;Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87:6378-6382; as well as U.S. Pat. Nos. 5,223,409, 5,198,346, and5,096,815).

Alternatively, other forms of mutagenesis can be utilized to generate acombinatorial library. For example, RAP-BP homologs (both agonist andantagonist forms) can be generated and isolated from a library generatedby using, for example, alanine scanning mutagenesis and the like (Ruf etal. (1994) Biochemistry 33:1565-1572; Wang et al. (1994)J. Biol. Chem.269:3095-3099; Balint et al. (1993) Gene 137:109-118; Grodberg et al.(1993) Eur. J Biochem. 218:597-601; Nagashima et al. (1993) J. Biol.Chem. 268:2888-2892; Lowman et al. (1991) Biochemistry 30:10832-10838;and Cunningham et al. (1989) Science 244:1081-1085), by linker scanningmutagenesis (Gustin et al. (1993) Virology 193:653-660; Brown et al.(1992) Mol. Cell. Biol. 12:2644-2652; McKnight et al. (1982) Science232:316); by saturation mutagenesis (Meyers et al. (1986) Science232:613); by PCR mutagenesis (Leung et al. (1989)Method Cell Mol Biol1:11-19); or by random mutagenesis (Miller et al. (1992) A Short Coursein Bacterial Genetics, CSHL Press, Cold Spring Harbor, N.Y.; and Greeneret al. (1994) Strategies in Mol Biol 7:32-34).

A wide range of techniques are known in the art for screening geneproducts of variegated gene libraries made by combinatorial mutagenesis,especially for identifying individual gene products having a certainproperty. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of, for example, RAPT1 homologs. The most widely usedtechniques for screening large gene libraries typically comprisescloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene whose product was detected. Each of theillustrative assays described below are amenable to high through-putanalysis as necessary to screen large numbers of degenerate RAP-BPsequences created by combinatorial mutagenesis techniques.

In one screening assay, the candidate RAP-BP gene products are displayedon the surface of a cell or viral particle, and the ability ofparticular cells or viral particles to bind the FKBP12/rapamycin complexvia this gene product is detected in a “panning assay”. For instance,the degenerate RAP-BP gene library can be cloned into the gene for asurface membrane protein of a bacterial cell, and the resulting fusionprotein detected by panning protocols (see, for example, Ladner et al.,WO 88/06630; Fuchs et al. (1991) Bio/Technology 9:1370-1371; and Gowardet al. (1992) TIBS 18:136-140). In a similar fashion, fluorescentlylabeled molecules which bind the RAP-binding protein, such asfluorescently labeled rapamycin or FKBP12/rapamycin complexes, can beused to score for potentially functional RAP-BP homologs. Cells can bevisually inspected and separated under a fluorescence microscope, or,where the morphology of the cell permits, separated by afluorescence-activated cell sorter.

In an alternate embodiment, the gene library is expressed as a fusionprotein on the surface of a viral particle. For instance, in thefilamentous phage system, foreign peptide sequences can be expressed onthe surface of infectious phage, thereby conferring two significantbenefits. First, since these phage can be applied to affinity matricesat very high concentrations, a large number of phage can be screened atone time. Second, since each infectious phage displays the combinatorialgene product on its surface, if a particular phage is recovered from anaffinity matrix in low yield, the phage can be amplified by anotherround of infection. The group of almost identical E. coli filamentousphages M13, fd, and fl are most often used in phage display libraries,as either of the phage gIII or gVIII coat proteins can be used togenerate fusion proteins without disrupting the ultimate packaging ofthe viral particle (Ladner et al. PCT publication WO-90/02909; Garrardet al., PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem.267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson etal. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS89:4457-4461). In an illustrative embodiment, the recombinant phageantibody system (RPAS, Pharmacia Catalog number 27-9400-01) can beeasily modified for use in expressing and screening RAP-BP combinatoriallibraries, and the RAP-BP phage library can be panned onglutathione-immobilized FKBP-GST/rapamycin complexes. Successive roundsof reinfection, phage amplification, and panning will greatly enrich forhomologs which retain FKBP/rapamycin binding and which can besubsequently screened for further biological activities in order todiscern between agonists and antagonists.

Homologs of the human and mouse RAP-binding proteins can also begenerated through the use of interaction trap assays to screencombinatorial libraries of RAP-BP mutants. As described in Example 10below, the same two hybrid assay used to screen cDNA libraries forproteins which interact with FK506-binding proteins in a drug-dependentmanner can also be used to sort through combinatorial libraries of, forexample, RAPT1 mutants, to find both agonistic and antagonistic forms.By controlling the sensitivity of the assay for interactions, e.g.through the manipulation of the strength of the promoter sequence usedto drive expression of the reporter construct, the assay can begenerated to favor agonistic forms of RAPT1 with tighter bindingaffinities for rapamycin then the authentic form of the protein.Alternatively, as described in Example 10, the assay can be used toselect for RAPT1 homologs which are now unable to bind rapamycincomplexes and hence are versions of the RAPT1 protein which can render acell insensitive to treatment with that macrolide.

The invention also provides for reduction of the rapamycin-bindingdomains of the subject RAP-binding proteins to generate mimetics, e.g.peptide or non-peptide agents, which are able to disrupt binding of apolypeptide of the present invention with an FKBP/rapamycin complex.Thus, such mutagenic techniques as described above are also useful tomap the determinants of RAP-binding proteins which participate ininteractions involved in, for example, binding to an FKBP/rapamycincomplex. To illustrate, the critical residues of a RAP-binding proteinwhich are involved in molecular recognition of FKBP/rapamycin can bedetermined and used to generate RAP-BP-derived peptidomimetics thatcompetitively inhibit binding of the RAP-BP to rapamycin complexes. Byemploying, for example, scanning mutagenesis to map the amino acidresidues of a particular RAP-binding protein involved in bindingFKBP/rapamycin complexes, peptidomimetic compounds can be generatedwhich mimic those residues in binding to the rapamycin complex, andwhich, by inhibiting binding of the RAP-BP to FKBP/rapamycin, caninterfere with the function of rapamycin in cell-cycle arrest. Forinstance, non-hydrolyzable peptide analogs of such residues can begenerated using retro-inverse peptides (e.g., see U.S. Pat. Nos.5,116,947 and 5,218,089; and Pallai et at (1983) Int J Pept Protein Res21:84-92) benzodiazepine (e.g., see Freidinger et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), azepine (e.g., see Huffman et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988), substituted gama lactam rings (Garvey et al. inPeptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher:Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson etal. (1986) J Med Chem 29:295; and Ewenson et al. in Peptides: Structureand Function (Proceedings of the 9th American Peptide Symposium) PierceChemical Co. Rockland, Ill., 1985), β-turn dipeptide cores (Nagai et al.(1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc PerkinTrans 1:1231), and β-aminoalcohols (Gordon et al. (1985) Biochem BiophysRes Commun 126:419; and Dann et al. (1986) Biochem Biophys Res Commun134:71). Utilizing side-by-side assays, peptidomimetics can be designedto specifically inhibit the interaction of human RAPT1 (or othermammalian homologs) with the FKBP12/rapamycin complex in mammaliancells, but which do not substantially affect the interaction of theyeast protein TOR1 or TOR2 with the FKB1/rapamycin complex. Such apeptide analog could be used in conjunction with rapamycin treatment ofmycotic infections to protect the host mammal from rapamycinside-effects, such as immunosuppression, without substantially reducingthe efficacy of rapamycin as an anti-fungal agent.

Another aspect of the invention pertains to an antibody specificallyreactive with one or more of the subject RAP-binding proteins. Forexample, by using immunogens derived from a RAP-binding protein,anti-protein/anti-peptide antisera or monoclonal antibodies can be madeby standard protocols (See, for example, Antibodies: A Laboratory Manualed. by Harlow and Lane (Cold Spring Harbor Press: 1988)). A mammal, suchas a mouse, a hamster or rabbit can be immunized with an immunogenicform of the peptide (e.g., a full length RAP-binding protein or anantigenic fragment which is capable of eliciting an antibody response).Techniques for conferring immunogenicity on a protein or peptide includeconjugation to carriers or other techniques well known in the art. Animmunogenic portion of the subject RAP-binding proteins can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassays can be used with the immunogen asantigen to assess the levels of antibodies. In a preferred embodiment,the subject antibodies are immunospecific for antigenic determinants ofthe RAP-binding proteins of the present invention, e.g. antigenicdeterminants of a protein represented in one of SEQ ID Nos: 2, 12 or 24or a closely related human or non-human mammalian homolog thereof. Forinstance, a favored anti-RAP-BP antibody of the present invention doesnot substantially cross react (i.e. react specifically) with a proteinwhich is less than 90 percent homologous to one of SEQ ID Nos: 2, 12 or24; though antibodies which do not substantially cross react with aprotein which is less than 95 percent homologous with one of SEQ ID Nos:2, 12 or 24, or even less than 98-99 percent homologous with one of SEQID Nos: 2; 12 or 24, are specifically contemplated. By “notsubstantially cross react”, it is meant that the antibody has a bindingaffinity for a non-homologous protein (e.g. a yeast TOR1 or TOR2protein) which is less than 10 percent, more preferably less than 5percent, and even more preferably less than 1 percent, of the bindingaffinity for a protein represented one of SEQ ID Nos: 2, 12 or 24.

Following immunization, anti-RAP-BP antisera can be obtained and, ifdesired, polyclonal anti-RAP-BP antibodies isolated from the serum. Toproduce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from an immunized animal and fused by standard somaticcell fusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, aninclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with a RAP-bindingprotein of the present invention and monoclonal antibodies isolated froma culture comprising such hybridoma cells.

An antibody preparation of this invention prepared from a polypeptide asdescribed above can be in dry form as obtained by lyophilization.However, the antibodies are normally used and supplied in an aqueousliquid composition in serum or a suitable buffer such as PBS.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with one of the subjectRAP-binding protein. Antibodies can be fragmented using conventionaltechniques, including recombinant engineering, and the fragmentsscreened for utility in the same manner as described above for wholeantibodies. For example, F(ab′)₂ fragments can be generated by treatingantibody with pepsin. The resulting F(ab′)₂ fragment can be treated toreduce disulfide bridges to produce Fab′ fragments. The antibody of thepresent invention is further intended to include bispecific and chimericmolecules having an anti-RAP-BP portion.

Both monoclonal and polyclonal antibodies (Ab) directed against aRAP-binding protein can be used to block the action of that protein andallow the study of the role of a particular RAP-binding protein in, forexample, cell-cycle regulation generally, or in the etiology ofproliferative and/or differentiative disorders specifically, or in themechanism of action of rapamycin, e.g. by microinjection of anti-RAP-BPantibodies into cells.

Antibodies which specifically bind RAP-BP epitopes can also be used inimmunohistochemical staining of tissue samples in order to evaluate theabundance and pattern of expression of each of the subject RAP-bindingproteins. Anti-RAP-BP antibodies can be used diagnostically inimmuno-precipitation and immuno-blotting to detect and evaluate RAP-BPlevels in tissue or bodily fluid as part of a clinical testingprocedure. For instance, such measurements as the level of free RAP-BPto RAP-BP/FKBP/drug complexes can be useful in predictive valuations ofthe efficacy of a particular rapamycin analog, and can permitdetermination of the efficacy of a given treatment regimen for anindividual. The level of a RAP-binding protein can be measured in cellsfound in bodily fluid, such as in cells from samples of blood, or can bemeasured in tissue, such as produced by biopsy.

Another application of the subject antibodies is in the immunologicalscreening of cDNA libraries constructed in expression vectors such asλgt11, λgt18-23, λZAP, and λORF8. Messenger libraries of this type,having coding sequences inserted in the correct reading frame andorientation, can produce fusion proteins. For instance, λgt11 willproduce fusion proteins whose amino termini consist of β-galactosidaseamino acid sequences and whose carboxy termini consist of a foreignpolypeptide. Antigenic epitopes of a RAP-binding protein can then bedetected with antibodies, as, for example, reacting nitrocellulosefilters lifted from infected plates with anti-RAP-BP antibodies. Phage,scored by this assay, can then be isolated from the infected plate.Thus, the presence of RAP-BP homologs can be detected and cloned fromother animals, and alternate isoforms (including splicing variants) canbe detected and cloned from human sources.

Moreover, the nucleotide sequence determined from the cloning of thesubject RAP-binding proteins from a human cell line will further allowfor the generation of probes designed for use in identifying homologs inother human cell types, as well as RAP-BP homologs (e.g. orthologs) fromother mammals. For example, by identifying highly conserved nucleotidessequence through comparison of the mammalian RAPT1 genes with the yeastTOR genes, it will be possible to design degenerate primers forisolating RAPT1 homologs from virtually any eukaryotic cell. Forinstance, alignment of the mouse RAPT1 gene sequence and the yeast DRR-1and TOR2 sequences, we have determined that optimal primers forisolating RAPT1 homologs from other mammalian homologs, as well as frompathogenic fungi, include the primers GRGAYTTRAWBGABGCHYAMGAWTGG,CAAGCBTGGGAYMTYMTYTAYTATMAYGTBTTCAG, and GAYYBGARTTGGCTG-TBCCHGG.

Accordingly, the present invention also provides a probe/primercomprising a substantially purified oligonucleotide, whicholigonucleotide comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least 10 consecutivenucleotides of sense or anti-sense sequence of one of SEQ ID Nos: 1, 11or 23, or naturally occurring mutants thereof. In preferred embodiments,the probe/primer further comprises a label group attached thereto andable to be detected, e.g. the label group is selected from the groupconsisting of radioisotopes, fluorescent compounds, enzymes, and enzymeco-factors. Such probes can also be used as a part of a diagnostic testkit for identifying transformed cells, such as for measuring a level ofa RAP-BP nucleic acid in a sample of cells from a patient; e.g.detecting mRNA encoding a RAP-BP mRNA level; e.g. determining whether agenomic RAP-BP gene has been mutated or deleted.

In addition, nucleotide probes can be generated which allow forhistological screening of intact tissue and tissue samples for thepresence of a RAP-BP mRNA. Similar to the diagnostic uses of anti-RAP-BPantibodies, the use of probes directed to RAP-BP mRNAs, or to genomicRAP-BP sequences, can be used for both predictive and therapeuticevaluation of allelic mutations which might be manifest in, for example,neoplastic or hyperplastic disorders (e.g. unwanted cell growth) orabnormal differentiation of tissue. Used in conjunction with an antibodyimmunoassays, the nucleotide probes can help facilitate thedetermination of the molecular basis for a developmental disorder whichmay involve some abnormality associated with expression (or lackthereof) of a RAP-binding protein. For instance, variation in synthesisof a RAP-binding protein can be distinguished from a mutation in thegenes coding sequence.

Thus, the present invention provides a method for determining if asubject is at risk for a disorder characterized by unwanted cellproliferation or abherent control of differentiation. In preferredembodiments, the subject method can be generally characterized ascomprising detecting, in a tissue sample of the subject (e.g. a humanpatient), the presence or absence of a genetic lesion characterized byat least one of (i) a mutation of a gene encoding one of the subjectRAP-binding proteins or (ii) the mis-expression of a RAP-BP gene. Toillustrate, such genetic lesions can be detected by ascertaining theexistence of at least one of (i) a deletion of one or more nucleotidesfrom a RAP-BP gene, (ii) an addition of one or more nucleotides to sucha RAP-BP gene, (iii) a substitution of one or more nucleotides of aRAP-BP gene, (iv) a gross chromosomal rearrangement of one of the RAP-BPgenes, (v) a gross alteration in the level of a messenger RNA transcriptof a RAP-BP gene, (vi) the presence of a non-wild type splicing patternof a messenger RNA transcript of a RAP-BP gene, and (vii) a non-wildtype level of a RAP-binding protein. In one aspect of the inventionthere is provided a probe/primer comprising an oligonucleotidecontaining a region of nucleotide sequence which is capable ofhybridizing to a sense or antisense sequence of one of SEQ ID Nos: 1, 11or 23, or naturally occurring mutants thereof, or 5′ or 3′ flankingsequences or intronic sequences naturally associated with the subjectRAP-BP genes. The probe is exposed to nucleic acid of a tissue sample;and the hybridization of the probe to the sample nucleic acid isdetected. In certain embodiments, detection of the lesion comprisesutilizing the probe/primer in a polymerase chain reaction (PCR) (see,e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202) or, alternatively, in aligation chain reaction (LCR) (see, e.g., Landegran et al. (1988)Science, 241:1077-1080; and NaKazawa et al. (1944) PNAS 91:360-364) thelater of which can be particularly useful for detecting point mutationsin the RAP-BP gene. Alternatively, immunoassays can be employed todetermine the level of RAP-binding protein and/or its participation inprotein complexes, particularly transcriptional regulatory complexessuch as those involving FKBP/rapamycin.

Also, by inhibiting endogenous production of a particular RAP-bindingprotein, anti-sense techniques (e.g. microinjection of antisensemolecules, or transfection with plasmids whose transcripts areanti-sense with regard to a RAP-BP mRNA or gene sequence) can be used toinvestigate role of each of the subject RAP-BP in growth anddifferentiative events, such as those giving rise to Wilm's tumor, aswell as normal cellular functions of each of the subject RAP-bindingproteins, e.g. in regulation of transcription. Such techniques can beutilized in cell culture, but can also be used in the creation oftransgenic animals.

Furthermore, by making available purified and recombinant RAP-bindingproteins, the present invention provides for the generation of assayswhich can be used to screen for drugs which are either agonists orantagonists of the cellular function of each of the subject RAP-bindingproteins, or of their role in the pathogenesis of proliferative anddifferentiative disorders. For instance, an assay can be generatedaccording to the present invention which evaluates the ability of acompound to modulate binding between a RAP-binding protein and anFK506-binding protein. In particular, such assays can be used to designand screen novel rapamycin analogs, as well as test completely unrelatedcompounds for their ability to mediate formation of FKBP/RAP-BPcomplexes. Such assays can be used to generate more potentanti-proliferative agents having a similar mechanism of action asrapamycin, e.g. rapamycin analogs. A variety of assay formats willsuffice and, in light of the present inventions, will be comprehended byskilled artisan.

One aspect of the present invention which facilitates the generation ofdrug screening assays, particularly the high-throughout assays describedbelow, is the identification of the rapamycin binding domain ofRAPT1-like proteins. For instance, the present invention providesportions of the RAPT1-like proteins which are easier to manipulate thanthe full length protein. The full length protein is, because of itssize, more difficult to express as a recombinant protein or a fusionprotein which would retain rapamycin-binding activity, and may very wellbe insoluble. Accordingly, the present invention provides solublepolypeptides which include a soluble portion of a RAPT1-like polypeptidethat binds to said FKBP/rapamycin complex, such as the rapamycin-bindingdomain represented by an amino acid sequence selected from the groupconsisting Val26-Tyr160 of SEQ ID No. 2, Val1272-Tyr1444 of SEQ ID No.12, Val41-Tyr173 of SEQ ID No. 14, Val1-Tyr133 of SEQ ID No. 16, andVal1-Arg133 of SEQ ID No. 18.

For instance, RAPT1 polypeptides useful in the subject screening assaysmay be represented by the general formula X-Y-Z, Y represents an aminoacid sequence of a rapamycin-binding domain within residues 1272 to 1444of SEQ ID No. 12, X is absent, or represents all or a C-terminal portionof the amino acid sequence between residues 1000 and 1444 of SEQ ID No.12 not represented by Y, and Z is absent, or represents all or aN-terminal portion of the amino acid sequence between residues 1272 and1809 of SEQ ID No. 12 not represented by Y. Preferably, the polypeptideincludes only about 50 to 200 residues of RAPT1 protein sequence.Similar polypeptides can be generated for other RAPT1-like proteins.

Moreover, the same formula can also be used to designate a fusionprotein, wherein Y represents a rapamycin-binding domain within residues1272 to 1444 of SEQ ID No. 12, X is absent or represents a polypeptidefrom 1 to about 500 amino acid residues of SEQ ID No. 12 immediatelyN-terminal to the rapamycin-binding domain, and Z is absent orrepresents from 1 to about 365 amino acid residues of SEQ ID No. 2immediately C-terminal to the rapamycin-binding domain.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target when contacted with a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity with other proteinsor change in enzymatic properties of the molecular target. Accordingly,in an exemplary screening assay of the present invention, the compoundof interest (the “drug”) is contacted with a mixture generated from anisolated and purified RAP-binding protein, such as RAPT1 or rapUBC, andan FK506-binding protein. Detection and quantification of drug-dependentFKBP/RAP-BP complexes provides a means for determining the compound'sefficacy for mediating complex formation between the two proteins. Theefficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide abaseline for comparison. In the control assay, isolated and purifiedRAP-BP is added to a composition containing the FK506-binding protein,and the formation of FKBPRAP-BP complexes is quantitated in the absenceof the test compound.

Complex formation between the RAP-binding protein and an FKBP/drugcomplex may be detected by a variety of techniques. For instance,modulation in the formation of complexes can be quantitated using, forexample, detectably labelled proteins (e.g. radiolabelled, fluorescentlylabelled, or enzymatically labelled), by immunoassay, or bychromatographic detection.

Typically, it will be desirable to immobilize either the FK506-bindingprotein or the RAP-binding protein to facilitate separation ofdrug-dependent protein complexes from uncomplexed forms of one of theproteins, as well as to accommodate automation of the assay. In anillustrative embodiment, a fusion protein can be provided which adds adomain that permits the protein to be bound to an insoluble matrix. Forexample, glutathione-S-transferase/FKBP (FKBP-GST) fusion proteins canbe adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the RAP-binding protein, e.g. an ³⁵S-labeled RAP-bindingprotein, and the test compound and incubated under conditions conduciveto complex formation (see, for instance, Example 9). Followingincubation, the beads are washed to remove any unbound RAP-BP, and thematrix bead-bound radiolabel determined directly (e.g. beads placed inscintillant), or in the supernatant after the FKBP/RAP-BP complexes aredissociated, e.g. when microtitre plates are used. Alternatively, afterwashing away unbound protein, the complexes can be dissociated from thematrix, separated by SDS-PAGE gel, and the level of RAP-BP found in thematrix-bound fraction quantitated from the gel using standardelectrophoretic techniques.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, the FK506-bindingprotein can be immobilized utilizing conjugation of biotin andstreptavidin. Biotinylated FKBP can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with the FKBP can be derivatized tothe wells of the plate, and FKBP trapped in the wells by antibodyconjugation. As above, preparations of a RAP-binding protein and a testcompound are incubated in the FKBP-presenting wells of the plate, andthe amount of FKBP/RAP-BP complex trapped in the well can bequantitated. Exemplary methods for detecting such complexes, in additionto those described above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with theRAP-binding protein, or which are reactive with the FK506-bindingprotein and compete for binding with the RAP-BP; as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the RAP-binding protein. In the instance of the latter,the enzymatic activity can be endogenous, such as a kinase (RAPT1) orubiquitin ligase (rapUBC) activity, or can be an exogenous activitychemically conjugated or provided as a fusion protein with theRAP-binding protein. To illustrate, the RAP-binding protein can bechemically cross-linked with alkaline phosphatase, and the amount ofRAP-BP trapped in the complex can be assessed with a chromogenicsubstrate of the enzyme, e.g. paranitrophenyl phosphate. Likewise, afusion protein comprising the RAP-BP and glutathione-S-transferase canbe provided, and complex formation quantitated by detecting the GSTactivity using 1-chloro-2,4-dinitrobenzene (Habig et al (1974) J BiolChem 249:7130).

For processes which rely on immunodetection for quantitating one of theproteins trapped in the complex, antibodies against the protein, such asthe anti-RAP-BP antibodies described herein, can be used. Alternatively,the protein to be detected in the complex can be “epitope tagged” in theform of a fusion protein which includes, in addition to the RAP-BP orFKBP sequence, a second polypeptide for which antibodies are readilyavailable (e.g. from commercial sources). For instance, the GST fusionproteins described above can also be used for quantification of bindingusing antibodies against the GST moiety. Other useful epitope tagsinclude myc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem266:21150-21157) which includes a 10-residue sequence from c-myc, aswell as the pFLAG system (International Biotechnologies, Inc.) or thepEZZ-protein A system (Pharamacia, N.J.).

Additionally, the subject RAP-binding proteins can be used to generate adrug-dependent interaction trap assay, as described in the examplesbelow, for detecting agents which induce complex formation between aRAP-binding protein and an FK506-binding protein. As described below,the interaction trap assay relies on reconstituting in vivo a functionaltranscriptional activator protein from two separate fusion proteins, oneof which comprises the DNA-binding domain of a transcriptional activatorfused to an FK506-binding protein (see also U.S. Pat. No. 5,283,317; PCTpublication WO94/10300; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; and Iwabuchi et al. (1993) Oncogene8:1693-1696). The second fusion protein comprises a transcriptionalactivation domain (e.g. able to initiate RNA polymerase transcription)fused to one of the subject RAP-binding proteins. When the FKBP andRAP-binding protein interact in the presence of an agent such asrapamycin, the two domains of the transcriptional activator protein arebrought into sufficient proximity as to cause transcription of areporter gene. In addition to the LexA interaction trap described in theexamples below, yet another illustrative embodiment comprisesSaccharomyces cerevisiae YPB2 cells transformed simultaneously with aplasmid encoding a GAL4 db-FKBP fusion (db: DNA binding domain) and witha plasmid encoding the GAL4 activation domain (GAL4ad) fused to asubject RAP-BP. Moreover, the strain is transformed such that theGAL4-responsive promoter drives expression of a phenotypic marker. Forexample, the ability to grow in the absence of histidine can depends onthe expression of the HIS3 gene. When the HIS3 gene is placed under thecontrol of a GAL4-responsive promoter, relief of this auxotrophicphenotype indicates that a functional GAL4 activator has beenreconstituted through the drug-dependent interaction of FKBP and theRAP-BP. Thus, agent able to promote RAP-BP interaction with an FKBP willresult in yeast cells able to grow in the absence of histidine.Commercial kits which can be modified to develop two-hybrid assays withthe subject RAP-binding proteins are presently available (e.g.,MATCHMAKER kit, ClonTech catalog number K1605-1, Palo Alto, Calif.).

In a preferred embodiment, assays which employ the subject mammalianRAP-binding proteins can be used to identify rapamycin mimetics thathave therapeutic indexes more favorable than rapamycin. For instance,rapamycin-like drugs can be identified by the present invention whichhave enhanced tissue-type or cell-type specificity relative torapamycin. To illustrate, the subject assays can be used to generatecompounds which preferentially inhibit IL-2 mediatedproliferation/activation of lymphocytes without substantiallyinterfering with other tissues, e.g. hepatocytes. Likewise, similarassays can be used to identify rapamycin-like drugs which inhibitproliferation of yeast cells or other lower eukaryotes, but which have asubstantially reduced effect on mammalian cells, thereby improvingtherapeutic index of the drug as an anti-mycotic agent relative torapamycin.

In one embodiment, the identification of such compounds is made possibleby the use of differential screening assays which detect and comparedrug-mediated formation of two or more different types of FKBP/RAP-BPcomplexes. To illustrate, the assay can be designed for side-by-sidecomparison of the effect of a test compound on the formation oftissue-type specific FKBP/RAPT1 complexes. Given the diversity of FKBPs,and the substantial likelihood that RAPT1 represents a single member ofa larger family of related proteins, it is probable that differentfunctional FKBP/RAPT1 complexes exist and, in certain instances, arelocalized to particular tissue or cell types. As described in PCTpublication WO93/23548, entitled “Method of Detecting Tissue-SpecificFK506 Binding Protein Messenger RNAs and Uses Thereof”, the tissuedistribution of FKBPs can vary from one species of the protein to thenext. Thus, test compounds can be screened for agents able to mediatethe tissue-specific formation of only a subset of the possiblerepertoire of FKBP/RAPT1 complexes. In an exemplary embodiment, aninteraction trap assay can be derived using two or more different baitproteins, e.g. FKBP12 (SEQ ID Nos. 5 and 6), FKBP25 (GenBank AccessionM90309), or FKBP52 (Genbank Accession M88279), while the fish protein isconstant in each, e.g. a human RAPT1 construct. Running the ITSside-by-side permits the detection of agents which have a greater effect(e.g. statistically significant on the formation of one of theFKBP/RAPT1 complexes than on the formation of the other FKBP complexes.

In similar fashion, differential screening assays can be used to exploitthe difference in drug-mediated formation of mammalian FKBP/RAP-BPcomplexes and yeast FKBP/TOR complexes in order to identify agents whichdisplay a statistically significant increase in specificity for theyeast complexes relative to the mammalian complexes. Thus, leadcompounds which act specifically on pathogens, such as fungus involvedin mycotic infections, can be developed. By way of illustration, thepresent assays can be used to screen for agents which may ultimately beuseful for inhibiting at least one fungus implicated in such mycosis ascandidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis,cryptococcosis, chromoblastomycosis, coccidioidomycosis,conidiosporosis, histoplasmosis, maduromycosis, rhinosporidosis,nocaidiosis, para-actinomycosis, penicilliosis, monoliasis, orsporotrichosis. For example, if the mycotic infection to which treatmentis desired is candidiasis, the present assay can comprise comparing therelative effectiveness of a test compound on mediating formation of amammalian FKBP/RAPT1 complex with its effectiveness towards mediatingsuch complexes formed from genes cloned from yeast selected from thegroup consisting of Candida albicans, Candida stellatoidea, Candidatropicalis, Candida parapsilosis, Candida krusei, Candidapseudotropicalis, Candida quillermondii, or Candida rugosa. Likewise,the present assay can be used to identify anti-fungal agents which mayhave therapeutic value in the treatment of aspergillosis by making useof the subject drug-dependent interaction trap assays derived from FKBPand TOR genes cloned from yeast such as Aspergillus fumigatus,Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, orAspergillus terreus. Where the mycotic infection is mucormycosis, thecomplexes can be derived from yeast such as Rhizopus arrhizus, Rhizopusoryzae, Absidia corymbifera, Absidia ramosa, or Mucor pusillus. Sourcesof other rapamycin dependent complexes for comparison with a mammalianFKBP/RAPT1 complex includes the pathogen Pneumocystis carinii. ExemplaryFK506-binding proteins from human pathogens and other lower eukaryotesare provided by, for example, GenBank Accession numbers: M84759 (Candidaalbican); U01195, U01198, U01197, U01193, U01188, U01194, U01199(Neisseria spp.); and M98428 (Streptomyces chrysomallus).

In an exemplary embodiment, the differential screening assay can begenerated using at least the rapamycin-binding domain of the Candidaalbican RAPT1 protein (see Example 11) and a Candida FK506-bindingprotein (such as RBP1, GenBank No. M84759, see also Ferrara et al.(1992) Gene 113:125-127), or a yeast FK506-binding protein (see Example8 and FIG. 3). Comparison of formation of human RAPT1 complexes andCandida RAPT1 complexes provides a means for identifying agents whichare more selective for the formation of caRAPT1 complexes and,accordingly, likely to be more specific as anti-mycotic agents relativeto rapamycin.

Furthermore, inhibitors of the enzymatic activity of each of the subjectRAP-binding proteins can be identified using assays derived frommeasuring the ability of an agent to inhibit catalytic conversion of asubstrate by the subject proteins. For example, the ability of thesubject RAPT1 proteins to phosphorylate a phosphatidylinositolsubstrate, such as phosphatidylinositol-4,5-biphosphate (PIP2), in thepresence and absence of a candidate inhibitor, can be determined usingstandard enzymatic assays. Likewise, the ability of the subjectubiquitin-conjugating enzyme to accept ubiquitin (e.g. from an E1:Ubconjugate) or subsequently transfer ubiquitin to a substrate protein,can be readily ascertained in the presence and absence of a candidateinhibitor. Exemplary assays in which the rapUBC enzyme of the presentinvention can be used are set forth in U.S. patent application Ser. No.08/176,937, entitled “Assay and Reagents for Detecting Inhibitors ofUbiquitin-dependent Degradation of Cell Cycle Regulatory Proteins”; thespecification of which was filed Jan. 4, 1994, and U.S. patentapplication Ser. No. 08/247,904, entitled “Human Ubiquitin ConjugatingEnzyme”, the specification of which was filed May 23, 1994.

Another aspect of the present invention concerns transgenic animalswhich are comprised of cells (of that animal) which contain a transgeneof the present invention and which preferably (though optionally)express an exogenous RAP-binding protein in one or more cells in theanimal. The RAP-BP transgene can encode the wild-type form of theprotein, or can encode homologs thereof, including both agonists andantagonists, as well as antisense constructs designed to inhibitexpression of the endogenous gene. In preferred embodiments, theexpression of the transgene is restricted to specific subsets of cells,tissues or developmental stages utilizing, for example, through the useof cis-acting sequences that control expression in the desired pattern.In the present invention, such mosaic expression of the subjectRAP-binding proteins can be essential for many forms of lineage analysisand can additionally provide a means to assess the effects ofloss-of-function mutations, which deficiency might grossly alterdevelopment in small patches of tissue within an otherwise normalembryo. Toward this and, tissue-specific regulatory sequences andconditional regulatory sequences can be used to control expression ofthe transgene in certain spatial patterns. Moreover, temporal patternsof expression can be provided by, for example, conditional recombinationsystems or prokaryotic transcriptional regulatory sequences.

Genetic techniques which allow for the expression of transgenes can beregulated via site-specific genetic manipulation in vivo are known tothose skilled in the art. For instance, genetic systems are availablewhich allow for the regulated expression of a recombinase that catalyzesthe genetic recombination a target sequence. As used herein, the phrase“target sequence” refers to a nucleotide sequence that is geneticallyrecombined by a recombinase. The target sequence is flanked byrecombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. Recombinase catalyzedrecombination events can be designed such that recombination of thetarget sequence results in either the activation or repression ofexpression of a subject RAP-binding protein. For example, excision of atarget sequence which interferes with the expression of a recombinantRAP-BP gene can be designed to activate expression of that gene. Thisinterference with expression of the protein can result from a variety ofmechanisms, such as spatial separation of the gene from a promoterelement or an internal stop codon. Moreover, the transgene can be madewherein the coding sequence of the gene is flanked by recombinaserecognition sequences and is initially transfected into cells in a 3′ to5′ orientation with respect to the promoter element. In such aninstance, inversion of the target sequence will reorient the subjectgene by placing the 5′ end of the coding sequence in an orientation withrespect to the promoter element which allow for promoter driventranscriptional activation.

In an illustrative embodiment, either the cre/loxP recombinase system ofbacteriophage P1 (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al.(1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomycescerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; PCTpublication WO 92/15694) can be used to generate in vivo site-specificgenetic recombination systems. Cre recombinase catalyzes thesite-specific recombination of an intervening target sequence locatedbetween loxP sequences. loxP sequences are 34 base pair nucleotiderepeat sequences to which the Cre recombinase binds and are required forCre recombinase mediated genetic recombination. The orientation of loxPsequences determines whether the intervening target sequence is excisedor inverted when Cre recombinase is present (Abremski et al. (1984) J.Biol. Chem. 259:1509-1514); catalyzing the excision of the targetsequence when the loxP sequences are oriented as direct repeats andcatalyzes inversion of the target sequence when loxP sequences areoriented as inverted repeats.

Accordingly, genetic recombination of the target sequence is dependenton expression of the Cre recombinase. Expression of the recombinase canbe regulated by promoter elements which are subject to regulatorycontrol, e.g., tissue-specific, developmental stage-specific, inducibleor repressible by externally added agents. This regulated control willresult in genetic recombination of the target sequence only in cellswhere recombinase expression is mediated by the promoter element. Thus,the activation expression of a RAP-binding protein can be regulated viaregulation of recombinase expression.

Use of the cre/loxP recombinase system to regulate expression of arecombinant RAP-binding protein, such as RAPT1 or rapUBC, requires theconstruction of a transgenic animal containing transgenes encoding boththe Cre recombinase and the subject protein. Animals containing both theCre recombinase and the recombinant RAP-BP genes can be provided throughthe construction of “double” transgenic animals. A convenient method forproviding such animals is to mate two transgenic animals each containinga transgene, e.g., the RAP-BP gene in one animal and recombinase gene inthe other.

One advantage derived from initially constructing transgenic animalscontaining a transgene in a recombinase-mediated expressible formatderives from the likelihood that the subject protein will be deleteriousupon expression in the transgenic animal. In such an instance, a founderpopulation, in which the subject transgene is silent in all tissues, canbe propagated and maintained. Individuals of this founder population canbe crossed with animals expressing the recombinase in, for example, oneor more tissues. Thus, the creation of a founder population in which,for example, an antagonistic RAP-BP transgene is silent will allow thestudy of progeny from that founder in which disruption of cell-cycleregulation in a particular tissue or at developmental stages wouldresult in, for example, a lethal phenotype.

Similar conditional transgenes can be provided using prokaryoticpromoter sequences which require prokaryotic proteins to be simultaneousexpressed in order to facilitate expression of the transgene. Exemplarypromoters and the corresponding trans-activating prokaryotic proteinsare given in U.S. Pat. No. 4,833,080. Moreover, expression of theconditional transgenes can be induced by gene therapy-like methodswherein a gene encoding the trans-activating protein, e.g. a recombinaseor a prokaryotic protein, is delivered to the tissue and caused to beexpressed using, for example, one of the gene therapy constructsdescribed above. By this method, the RAP-BP transgene could remainsilent into adulthood and its expression “turned on” by the introductionof the trans-activator.

In an exemplary embodiment, the “transgenic non-human animals” of theinvention are produced by introducing transgenes into the germline ofthe non-human animal. Embryonal target cells at various developmentalstages can be used to introduce transgenes. Different methods are useddepending on the stage of development of the embryonal target cell. Thezygote is the best target for micro-injection. In the mouse, the malepronucleus reaches the size of approximately 20 micrometers in diameterwhich allows reproducible injection of 1-2 pl of DNA solution. The useof zygotes as a target for gene transfer has a major advantage in thatin most cases the injected DNA will be incorporated into the host genebefore the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). Asa consequence, all cells of the transgenic non-human animal will carrythe incorporated transgene. This will in general also be reflected inthe efficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. Microinjection ofzygotes is the preferred method for incorporating transgenes inpracticing the invention.

Retroviral infection can also be used to introduce a RAP-BP transgeneinto a non-human animal. The developing non-human embryo can be culturedin vitro to the blastocyst stage. During this time, the blastomeres canbe targets for retroviral infection (Jaenich, R. (1976) PNAS73:1260-1264). Efficient infection of the blastomeres is obtained byenzymatic treatment to remove the zona pellucida (Manipulating the MouseEmbryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, 1986). The viral vector system used to introduce the transgeneis typically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152). Transfection is easily and efficiently obtained byculturing the blastomeres on a monolayer of virus-producing cells (Vander Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).Alternatively, infection can be performed at a later stage. Virus orvirus-producing cells can be injected into the blastocoele (Jahner etal. (1982) Nature 298:623-628). Most of the founders will be mosaic forthe transgene since incorporation occurs only in a subset of the cellswhich formed the transgenic non-human animal. Further, the founder maycontain various retroviral insertions of the transgene at differentpositions in the genome which generally will segregate in the offspring.In addition, it is also possible to introduce transgenes into the germline by intrauterine retroviral infection of the midgestation embryo(Jahner et al. (1982) supra).

A third type of target cell for transgene introduction is the embryonalstem cell (ES). ES cells are obtained from pre-implantation embryoscultured in vitro and fused with embryos (Evans et al. (1981) Nature292:154-156; Bradley et al. (1984) Nature 309:255-258; Gossler et al.(1986) PNAS 83: 9065-9069; and Robertson et al. (1986) Nature322:445-448). Transgenes can be efficiently introduced into the ES cellsby DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal. For reviewsee Jaenisch, R. (1988) Science 240:1468-1474.

Methods of making knock-out or disruption transgenic animals are alsogenerally known. See, for example, Manipulating the Mouse Embryo, (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).Recombinase dependent knockouts can also be generated, e.g. byhomologous recombination to insert recombinase target sequences, suchthat tissue specific and/or temporal control of inactivation of a RAP-BPgene can be controlled as above.

Another aspect of the present invention concerns a novel in vivo methodfor the isolation of genes encoding proteins which physically interactwith a “bait” protein/drug complex. The method relies on detecting thereconstitution of a transcriptional activator in the presence of thedrug, particularly wherein the drug is a non-peptidyl small organicmolecule (e.g. <2500K), e.g. a macrolide, e.g. rapamycin, FK506 orcyclosporin. In particular, the method makes use of chimeric genes whichexpress hybrid proteins. The first hybrid comprises the DNA-bindingdomain of a transcriptional activator fused to the bait protein. Thesecond hybrid protein contains a transcriptional activation domain fusedto a “fish” protein, e.g. a test protein derived from a cDNA library. Ifthe fish and bait proteins are able to interact in a drug-dependentmanner, they bring into close proximity the two domains of thetranscriptional activator. This proximity is sufficient to causetranscription of a reporter gene which is operably linked to atranscriptional regulatory site responsive to the transcriptionalactivator, and expression of the marker gene can be detected and used toscore for the interaction of the bait protein/drug complex with anotherprotein.

One advantage of this method is that a multiplicity of proteins can, besimultaneously tested to determine whether any interact with thedrug/protein complex. For example, a DNA fragment encoding theDNA-binding domain can be fused to a DNA fragment encoding the baitprotein in order to provide one hybrid. This hybrid is introduced intothe cells carrying the marker gene, and the cells are contacted with adrug which is known to bind the bait protein. For the second hybrid, alibrary of plasmids can be constructed which may include, for example,total mammalian complementary DNA (cDNA) fused to the DNA sequenceencoding the activation domain. This library is introduced into thecells carrying the first hybrid. If any individual plasmid from the testlibrary encodes a protein that is capable of interacting with thedrug/protein complex, a positive signal may be obtained by detectingexpression of the reporter gene. In addition, when the interactionbetween the drug complex and a novel protein occurs, the gene for thenewly identified protein is readily available.

As illustrated herein, the present interaction trap system is a valuabletool in the identification of novel genes encoding proteins which act ata point in a given signal transduction pathway that is directly upstreamor downstream from a particular protein/drug complex. For example, thesubject assay can be used to identify the immediate downstream targetsof an FKBP/rapamycin complex, or of an FKBP/FK506 complex, or of acyclophilin/cyclosporin complex. Proteins that interact in adrug-dependent manner with one of such complexes may be identified, andthese proteins can be of both diagnostic and therapeutic value.

A first chimeric gene is provided which is capable of being expressed inthe host cell, preferably a yeast cell, most preferably Saccharomycescerevisiae or Schizosaccharomyces pombe. The host cell contains adetectable gene having a binding site for the DNA-binding domain of thetranscriptional activator, such that the gene expresses a marker proteinwhen the marker gene is transcriptionally activated. Such activationoccurs when the transcriptional activation domain of a transcriptionalactivator is brought into sufficient proximity to the DNA-binding domainof the transcriptional activator. The first chimeric gene may be presentin a chromosome of the host cell. The gene encodes a chimeric proteinwhich comprises a DNA-binding domain that recognizes the binding site onthe marker gene in the host cell and a bait protein which is to betested for drug-mediated interaction with a second test protein orprotein fragment.

A second chimeric gene is provided which is capable of being expressedin the host cell. In one embodiment, both the first and the secondchimeric genes are introduced into the host cell in the form ofplasmids. Preferably, however, the first chimeric gene is present in achromosome of the host cell and the second chimeric gene is introducedinto the host cell as part of a plasmid. The second chimeric genecontains a DNA sequence that encodes a second hybrid protein. The secondhybrid protein contains a transcriptional activation domain. The secondhybrid protein also contains a second test protein or a protein fragmentwhich is to be tested for interaction with the first test protein orprotein fragment. Preferably, the DNA-binding domain of the first hybridprotein and the transcriptional activation domain of the second hybridprotein are derived from transcriptional activators having separateDNA-binding and transcriptional activation domains. These separateDNA-binding and transcriptional activation domains are also known to befound in the yeast GAL4 protein, and are also known to be found in theyeast GCN4 and ADR1 proteins. Many other proteins involved intranscription also have separable binding and transcriptional activationdomains which make them useful for the present invention. In anotherembodiment, the DNA-binding domain and the transcriptional activationdomain may be from different transcriptional activators. The secondhybrid protein is preferably encoded on a library of plasmids thatcontain genomic, cDNA or synthetically generated DNA sequences fused tothe DNA sequence encoding the transcriptional activation domain.

The drug-mediated interaction between the first test protein and thesecond test protein in the host cell, therefore, causes thetranscriptional activation domain to activate transcription of thedetectable gene. The method is carried out by introducing the firstchimeric gene and the second chimeric gene into the host cell, andcontacting the cell with the drug of interest. The host cell issubjected to conditions under which the first hybrid protein and thesecond hybrid protein are expressed in sufficient quantity for thedetectable gene to be activated. The cells are then tested fordrug-dependent expression of the detectable gene.

Thus, interactions between a first test protein and a library ofproteins can be tested in the presence of the drug of interest, in orderto determine which members of the library are involved in the formationof drug-dependent complexes between the first and second protein. Forexample, the bait protein may be a protein which binds FK506, rapamycin,or cyclosporin, e.g. can be an FKBP or cyclophilin. The second testprotein may be derived from a cDNA library.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 Construction of the Bait Plasmids for the 2-Hybrid Screen A.LexA-FKBP12 Bait:

The bait protein and fish protein constructs used in the presentdrug-dependent interaction trap are essentially the same as constructsused for other 2 hybrid assays (see, for example, U.S. Pat. No.5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) JBiol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696). Using thefollowing oligonucleotides:

coding strand (SEQ ID No: 3)G GGT TTG GAA TTC CTA ATA ATG TCT GTA CAA GTA GAA ACC non-coding strand(SEQ ID No: 4) GGG TTT CGG GAT CCC GTC ATT CCA GTT TTA GAA GPCR amplification was carried out from a lymphocyte cDNA library toisolated the coding sequence for the FKBP12 protein. The sequence of thehuman FKBP12 cloned was confirmed as:

(SEQ ID No: 5) ATGTCCGTACAAGTAGAAACCATCTcCCCAGGAGACGGGCGCACCTTcCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGgATGCTTGAAGATGGAAAGAAATTTGATTCCTCCCGTGACCGTAACAAGCCCTTTAAGTTtATgCTAGGCaAGCAGGAGGTGATCCGAGGCTGGGAAGAagGGGTTGcCCAGATGAGTGTGGgTCAGCGTGCCAAaCTgACTAtAtCTCcAGaTtATgCcTATGgTGCCACTGGGCAccCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAATGA

The resulting PCR product containing the human FKBP12 coding sequenceswas then digested with EcoRI and BamHI, and cloned into the EcoRI+BamHIsites of pBTM116 creating an in-frame fusion between LexA and FKBP12.The resulting plasmid is referred to below as plC504.

B. LexA-(gly)₆-FKBP12 Bait:

In order to generate an in frame fusion between LexA and FKBP12separated by six glycine residues, the coding sequence from human FKBP12was cloned by PCR as above, except that the sense oligonucleotideprovided an additional 18 nucleotides which inserted 6 glycines in theopen reading frame of the fusion protein. The oligos used for PCR were:

coding strand (SEQ ID No: 7)TCG CCG GAA TTC GGG GGC GGA GGT GGA GGA GTA CAA GTA GAA ACC ATCnon-coding strand (SEQ ID No: 8)GGG TTT CGG GAT CCC GTC ATT CCA GTT TTA GAA G

The PCR product containing the human FKBP12 coding sequences was thendigested with EcoRI and BamHI and cloned into the EcoRI+BamHI sites ofpBTM116 as above. The resulting plasmid is referred to below as plC506.

Example 2 Construction of the FKBP12 Deletion Strain

A 1.8 kb HindIII-EcoRI yeast genomic fragment containing FKB1 (the S.Cerveisia homolog of FKBP12) was cloned into the HindIII+EcoRI sites ofpSP72 (Promega).

A one-step PCR strategy was used to create a precise deletion of the FKB1 coding sequences extending from the ATG start codon to the TGA stopcodon. Simultaneously a unique BamHI site was introduced in lieu of theFKB 1 coding sequences. The oligos used to generate the FKB1 deletionand introduction of the unique BamHI site were:

(SEQ ID No: 9) CGCGGATCCGCGCATTATTACTTGTTTTGATTGATTTTTTG (SEQ ID No: 10)CGCGGATCCGCGTAAAAGCAAAGTACTATCAATTGAGCCG

The yeast ADE2 gene on a 3.6 kb BamHI fragment was then cloned into theunique BamHI site of the plasmid described above to generate the plasmidpVB172. Flanking the ADE2 disruption marker of pVB172 in the 5′ and 3′noncoding sequence of FKB1 are XhoI sites. pVB172 was digested with XhoIto release a linear fragment containing ADE2 flanked by FKBI noncodingsequences. This linear fragment was used to transform yeast strain L40(Mat a his3 Δ200 trp1-901 leu2-3,112 ade2 LYS2::(lexAop)4-HIS3URA3::(lexAop)₈-lacZ GAL4 gal80) selecting for adenine prototrophy.

ADE+ yeast transformants were tested for rapamycin resistance to confirmthat the wild type FKB1 allele was replaced by ADE2. This disruptionallele of FKB1 is designated L40-fkb1-2.

Example 3 Cloning Of Mammalian Rapamycin Target Genes

We used the drug-dependent interaction trap described in Example 1above, with the LexA binding-domain fusion constructs as bait to detectinteraction with clones from cDNA libraries containing VP16activation-domain fusions. The reporters used as “read-outs” signalinginteraction in this system are the S. cerevisiae HIS3 and the E. coliLacZ genes. The yeast strain L40, the bait vector plasmid pBTM116 andthe mouse embryonic PCR library in the vector pVP16 were used toconstruct the cDNA fusion protein library

The strain L40-fkb1-2, described above in Example 2, was transformedwith each of two bait plasmids, plC504, encoding the LexA-FKBP12 fusionprotein, or p10506, encoding the LexA-(gly)6-FKBP12 fusion protein. Thetransformants, L40-fkb1-2/p10504 (named ICY99) and L40-fkb1-2/plC506(named ICY101) were maintained on yeast media lacking tryptophan whichselects for cells harboring the bait plasmid.

A mouse embryo PCR library in pVP16 (designated pSH10.5), which wasgenerated by standard protocols using random-primed synthesis of 10.5day-post-coital CD1 mouse embryo polyA+ RNA and size-selected forinserts between 350 bp and 700 bp in length, was used to transform theyeast ICY99 and ICY101. The transformed yeast cells were plated ontomedia lacking tryptophan and leucine. Approximately 10⁷ transformantsfrom each strain were pooled, thoroughly mixed, and stored frozen inaliquots in 50% glycerol at −80° C.

Prior to screening, cells were thawed, grown for 5 hours in liquidmedium, and plated onto selective medium. Approximately 1.5×10⁷ICY99/pSH10.5 cells were plated onto phosphate-buffered (pH7) syntheticagar medium containing (i) all amino acids except tryptophan, leucineand histidine, (ii) Rapamycin at 125 ng/ml, (iii) the chromogenicsubstrate X-gal at 100 ng/ml, and (iv) 2% glucose as carbon source, at aplating density of approximately 10⁶ per 15 cm plate. An identicalprotocol was used for screening ICY101/pSH10.5 transformants, exceptthat a lower concentration of rapamycin was used, at 15.6 ng/ml.

Colonies which both grew on the selective medium and were blue werepicked for further testing. These represent cells which do not requirehistidine for growth and which are expressing the β-galactosidasereporter. Candidate colonies appeared between 4-11 days after plating,and the blue color ranged from very light blue to deep blue. They werethen subjected to the following tests.

i) Rapamycin-Dependence

Each candidate was streaked onto media lacking histidine and containingeither 125 ng/ml (for ICY99/pSH10.5 candidates), 15.6 ng/ml (forICY101/pSH10.5 candidates) rapamycin, or no rapamycin (for both).Candidate clones which grew in the presence of rapamycin and failed togrow on media without rapamycin were chosen for the next test.

For the ICY99/pSH10.5 screen, out of 107 His+ and LacZ+ candidatesscreened, 24 were rapamycin-dependent for growth on medium lackinghistidine. For the ICY101/pSH10.5 screen, 20 out of 101 His+ and LacZ+candidates screened were rapamycin-dependent.

ii) Plasmid-Linkage

To eliminate false positives caused by chromosomal mutations, eachcandidate was grown in non-selective medium (YPD) to permit loss of thebait (Trp+) and the cDNA (Leu+) plasmids. Cells which had lost the baitplasmid (Trp−), the cDNA plasmid (Leu−) or both plasmids (Trp− andLeu−), as well as those which had retained both plasmids (Trp+ andLeu+), were streaked onto media containing rapamycin but lackinghistidine. Those candidates for which only the derivatives containingboth plasmids (Trp+ and Leu+) grew, while the other three derivativesdid not, were chosen for further analysis.

For the ICY99/pSH10.5 screen, 23 out of 24 passed the test. For theICY101/pSH10.5 screen, all 20 passed the test.

iii) Positive and Negative Interaction with Control Baits

Whereas the previous test asked if the interaction disappears wheneither or both members of the interaction (bait and fish constructs) arelost, the present test asks if the candidate cDNA plasmid (Leu+) canconfer interaction when transformed into yeast strains harboring variousbaits. DNA samples were prepared from each candidate and used totransform E. coli strain B290 (auxotrophic for tryptophan and leucine).Since the yeast TRP1 and LEU2 genes can complement the bacterialauxotrophies, respectively, B290 cells containing the bait plasmid areTrp+ and can grow on medium lacking tryptophan, while B290 cellscontaining the cDNA plasmid are Leu+ and can grow on medium lackingleucine. Plasmid DNA samples were each containing a different bait: i)ICY99, the original strain used in the screen, containing theLexA::FKBP12 bait fusion; ii) ICY101, containing theLexA::(gly)₆::FKBP12 bait fusion, and iii) ICY102, containing a LexAfusion bait irrelevant for the present study and which serves as anegative control. The ideal candidate clone should confer His+ and LacZ+to ICY99 and ICY101 in a rapamycin-dependent manner, but not to ICY102.

For the ICY99/pSH10.5 screen, 11 out of the 23 candidates fulfilled theabove criteria. For the ICY101/pSH10.5 screen, 10 out of the 20candidates fulfilled the above criteria.

The cDNA inserts of these candidate clones were sequenced in bothstrands using the ABI fluorescent sequencing system. All 11 candidatesfrom the ICY99/pSH10.5 screen, and at least 4 out of 10 of thecandidates from the ICY101/pSH10.5 screen contain overlapping fragmentsof an identical sequence. The 14 clones represent at least 5 independentcloning events from the library as judged by the insert/vectorboundaries of each clone. The longest and the shortest inserts differ byapproximately 70 bp at the amino-terminus and about 10 bp at theamino-terminus. The partial nucleotide sequence, and corresponding aminoacid sequence, isolated from the mouse rapamycin/FKBP 12 binding protein(RAPT1), is given in SEQ ID No: 1 and SEQ ID No: 2, respectively.

Surprisingly, a search of the GenBank database using the program BLAST,revealed that the peptide encoded by the above sequence shares somehomology, though less than 60 percent absolute homology, to the S.cerevisiae TOR1 (and DRR1) and TOR2 gene products previously isolatedfrom yeast.

Example 4 Cloning of Human Homologs of Rapamycin Target Genes

Having isolated a partial sequence for the gene encoding arapamycin-target-protein from a mouse library, we proceeded to isolatethe human gene using the mouse sequence as a probe. The plasmid cloneplC99.1.5, containing the longest insert of the RAPT1 clone, was chosenas probe for hybridization. The insert (500 bp) was separated fromplasmid DNA by digestion with Not I restriction endonuclease followed byagarose gel electrophoresis and fragment purification. The fragment wasradiolabelled with αP³²-labeled dCTP by random-incorporation with theKlenow fragment of DNA polymerase. The radiolabelled DNA probe wasisolated away from free nucleotides by a G50 column, alkali-denatured,and added to the hybridization mix at 2×10⁶ cpm/ml.

Approximately 3×10⁶ phage of a human B cell cDNA library in λ-pACT(FIG. 1) were screened by filter hybridization using the probe describedabove, in 30% formamide, 5×SSC, 5×Denhardts, 20 μg/ml denatured salmonsperm DNA, and 1% SDS, at 37° C. Following hybridization, the filterswere washed at 0.5×SSC and 0.1% SDS, at 50° C. These representconditions of medium stringency appropriate for mouse-to-humancross-species hybridizations. A number of positive plaques wereobtained, and several were analyzed. A number of the isolated clonesturned out to be various 3′ fragments of the same gene, or very closelyrelated genes, which, after sequence analysis, was determined to be thehuman RAPT1 gene. The clone containing the longest coding sequencefragment, comprising what is believed to be roughly half the full-lengthprotein (C-terminus) and including the FKBP/rapamycin binding site andthe putative PI-kinase activity, is designated as plasmid pIC524. Adeposit of the pACT plasmid form of pIC524 was made with the AmericanType Culture Collection (Rockville, Md.) on May 27, 1994, under theterms of the Budapest Treaty. ATCC Accession number 75787 has beenassigned to the deposit.

FIG. 1 is a map of the human RAPT1 clone of pIC524 (inserted at the XhoIsite). The insert is approximately 3.74 kb in length, and nucleotideRAPT1 coding sequence from the insert has been obtained and isrepresented by nucleotide residues 2401-5430 of SEQ ID No. 11. Thecorresponding amino acid sequence is represented by residuesHis801-Trp1809 of SEQ ID No. 12. The region of the human RAPT1 clonecorresponding to the mouse RAPT1 fragment is greater than 95% homologousat the amino acid level and 90% homologous at the nucleotide level. Inaddition to the pIC524 clone, further 5′ sequence of the human RAPT1gene was obtained from other overlapping clones, with the additionalsequence of the 3′ end of the ˜5.4 kb partial gene given in SEQ ID No.11. Furthermore, SEQ ID No. 19 provides additional 3′ non-codingsequence (obtained from another clone) which flanks the RAPT1 codingsequence.

It will be evident to those skilled in the art that, given the presentsequence information, PCR primers can be designed to amplify all, orcertain fragments of the RAPT1 gene sequence provided in pIC524. Forexample, the primers TGAAGATACCCCACCAA-ACCC (SEQ ID No. 21) andTGCACAGTTGAAGTGAAC (SEQ ID No. 22) correspond to pACT sequences flankingthe XhoI site, and can be used to PCR amplify the entire RAPT1 sequencefrom pIC524. Alternatively, primers based on the nucleic acid sequenceof SEQ ID No. 11 can be used to amplify fragments of the RAPT1 gene inpIC524. The PCR primers can be subsequently sub-cloned into expressionvectors, and used to produce recombinant forms of the subject RAPT1protein. Thus, the present provides recombinant RAPT1 proteins encodedby recombinant genes comprising RAPT1 nucleotide sequences from ATCCdeposit number 75787. Moreover, it is clear that primer/probes can begenerated which include even those portion of pIC524 not yet sequencedby simply providing PCR primers based on the known sequences.

Furthermore, our preliminary data indicate that other proteins which arerelated to RAPT1, e.g. RAPT1 homologs, were also obtained from thepresent assay, suggesting that RAPT1 is a member of a larger family ofrelated proteins.

Example 5 Cloning of Novel Human Ubiquitin Conjugating Enzyme

Constructs similar to those described above for the drug-dependentinteraction trap assay were used to screen a WI38 (mixed G₀ and dividingfibroblast) cDNA library (Clonetech, Palo Alto Calif.) in pGADGH (XhoIinsert, Clonetech). Briefly, the two hybrid assay was carried out asabove, using GAL4 constructs instead of LexA, and in an HF7C yeast cell(Clonetech) in which FKB1 gene was disrupted (see Example 1). Of theclones isolated, a novel human ubiquitin-conjugating enzyme (rap-UBC)has been identified. A deposit of the pGADGH plasmid (clone “SMR4-15”)was made with the American Type Culture Collection (Rockville, Md.) onMay 27, 1994, under the terms of the Budapest Treaty. ATCC Accessionnumber 75786 has been assigned to the deposit. The insert isapproximately 1 kB.

The sequence of the 5′ portion of the SMR4-15 insert is given by SEQ IDNo. 23 (nucleotide) and SEQ ID No. 24 (amino acid) and comprises asubstantial portion of the coding region for rap-UBC, including theactive site cysteine: The sequence for the 3′ portion of the clone isprovided by SEQ ID No. 25. As described above, primers based on thenucleic acid sequence of SEQ ID No. 23 (and 25) can be used to amplifyfragments of the rap-UBC gene from SMR4-15. The PCR primers can besubsequently sub-cloned into expression vectors, and used to producerecombinant forms of the subject enzyme. Thus, the present providesrecombinant rap-UBC proteins encoded by recombinant genes comprisingrap-UBC nucleotide sequences from ATCC deposit number 75786.

Example 6 Construction of the Serine-to-Argenine mRAPT1 Mutation

The smallest mRAPT1 clone that interacted with the FKBP12/rapamycincomplex was 399 bp, defingin a rapamycin binding domain. The RAPT1binding domain corresponds to a region in yeast TOR1/TOR2 locatedimmediately upstream, but outside of the lipid kinase consensussequence. This region contains the serine residue which when mutated inyeast TOR1 confers resistance to rapamycin (Cafferkey et al. (1993) MolCell Biol 13:6012-6023). A mouse RAPT1 serine-to-argenine mutation wasconstructed by oligonucleotide mutagenesis. Coding and noncoding strandoligonucleotides containing the mutations were: GAAGAGGCAAGACGCTTGTAC(SEQ ID NO:26) and GTACAAGCGTCTTGCC-TCTTC (SEQ ID NO:27). PCR reactionswere performed using these oligonucleotides in combination witholigonucleotides GAGTTTGAGCAGATGTTTA (SEQ ID NO:28) and the M13universal primer which are sequences in the pVP16 vector, 5′ and 3′ ofthe mRAPT1 insert, respectively. pVP16 containing mRAPT1 was used as thetemplate for PCR. The PCR product, digested with BamHI and EcoRI, wascloned into the BamHI and EcoRI sites in pVP16. The resulting clone wassequenced to verify that the clone contained the serine-to-argeninemutation and no others.

The smallest mRAPT1 clone that interacted with the FKBP12/rapamycincomplex was 399 bp, defining the RAPT1 binding domain. The RAPT1 bindingdomain corresponds to a region in yeast TOR located immediatelyupstream, but outside of the lipid kinase consensus sequence. Thisregion contains the serine residue which when mutated in yeast TOR1(also called DRR1) confers resistance to rapamycin (Cafferkey et al.(1993) Mol. Cell. Biol. 13:6012-6023; Helliwell et al. (1994) Mol. Cell.Biol. 5:105-118). The corresponding mutation was constructed in mRAPT1.The serine-to-argenine mutation abolishes interaction of mRAPT1 with theFKBP12/rapamycin complex (see FIG. 3), activating neither HIS3 nor lacZexpression on the two-hybrid assay, indicating that the serine isinvolved in the association of the FKBP12/rapamycin complex with mRAPT1.

Example 7 Northern Analysis

The multiple tissue Northern blots (containing 2 μg of human RNA perlane) were obtained from Clonetech Labs., Inc. Hybridizations were at42° C. in 5×SSPE, 5×Denhardt's, 30% formamide, 1% SDS and 200 μg/mldenatured salmon sperm DNA. Washes were at 0.1×SSC and 0.1% SDS at 55°C. The blot was exposed for 5 days prior to autoradiography. The levelsof RNA loaded in each lane were independently monitored by hybridizingthe same blots with a human G3PDH probe and were found to be similar inall lanes, with the exception of skeletal muscle, which hadapproximately 2-3 fold the signal.

RAPT1 specifies a single transcript of approximately 9 kb that ispresent in all tissues examined, exhibiting the highest levels intestis. The transcript is sufficient to encode a protein equivalent tothe size of yeast TOR which is 284 kDa. Assuming that RAPT1 is ofsimilar size, a small fragment of 133 amino acids has been cloned fromwithin a large protein, but which fragment is sufficient to bindFKBP12/rapamycin complex.

Example 8 High Throughput Assay Based on the Two-Hybrid System forIdentifying Novel Rapamycin Analogs

To develop a high throughput screen based on the two-hybrid system, wedevised a procedure to quantitate protein-protein interaction mediatedby a small molecule. Since protein-protein interaction in the two-hybridsystem stimulates transcription of the lacZ reporter gene, the assayutilizes a substrate of β-galactosidase (the lacZ gene product lacZ geneproduct) which when cleaved produces a chemiluminescent signal that canbe quantitated. This assay can be performed in microtiter plates,allowing thousands of compounds to be screened per week. The assayincludes the following steps:

-   1. Inoculate yeast cells from a single colony into 50 ml of growth    medium, synthetic complete medium lacking leucine and tryptophan    (Sherman, F. (1991) Methods Enzymol. 194:3-20). Incubate the flask    overnight at 30° C. with shaking (˜200 rpm).-   2. Dilute the overnight culture to a final A₆₀₀ of 0.02 in growth    medium and incubate overnight as described in step 1.-   3. Dilute the second overnight culture to a final A₆₀₀ of 0.5 in    growth medium. Using a Quadra 96 pipettor (TomTec, Inc.), dispense    135 μl aliquots of the cell suspension into wells of a round bottom    microtiter plate pre-loaded with 15 μl/well of the compound to be    tested at various concentrations. (The compounds are dissolved in 5%    dimethyl sulfoxide, so that the final DMSO concentration added to    cells is 0.5% which does not perturb yeast cell growth.) Cover    microtiter plates and incubate at 30° C. for 4 hr with shaking at    300 rpm.-   4. Centrifuge microtiter plate for 10 mM at 2000 rpm. Remove the    supernatant with the Quadra 96 pipettor and wash with 225 μl    phosphate buffered saline.-   5. Dispense 100 μl of lysis buffer (100 mM₂HPO₄ pH 7.8; 0.2% Triton    X-100; 1.0 mM ditiothriotol) into each well, cover, and incubate for    30 min at room temperature with shaking at 300 rpm.-   6. Dispense into each well of a Microfluor plate (Dynatech    Laboratories, Chantilly, Va.), 50 μl of the chemiluminescent    substrate, Galacton Plus™ (Tropix, Inc., Bedford, Mass.) in diluent    (100 mM Na₂HPO₄, 1 mM MgCl2, pH 8.0). To these wells, transfer 20 μl    of cell lysate and incubate in the dark for 60 min at room    temperature.-   7. Add to each well 75 μl of Emeral™ accelerator. Cover plate and    count in a Topcount scintillation counter (Packard, Inc.) for 0.01    min/well.

The rapamycin target proteins, isolated as described above, wereincorporated into the quantitative assay, as was a variety of FKBPs. TheFKBPs included in the screen were human FKBP12 and that from pathogenicfungi, FKBP13 (Jin et al. (1991) Proc. Natl. Acad. Sci. 88:6677) andFKBP25 (Jin et al. (1992) J. Biol. Chem. 267:2942; Galat et al. (1992)Biochem. 31:2427-2434). Yeast strains containing different FKBP-targetpairs can be tested against libraries of rapamycin and FK506 analogs.Such a screen can yield different classes of compounds including (i)target-specific compounds, those that mediate interaction between aspecific target and more than one FKBP, (ii) FKBP-specific compounds,those that mediate interaction between a particular FKBP and more thanone target and, most ideally, (iii) FKBP/target-specific compounds,those that mediate interaction between a particular FKBP and target. Theprotein interactions mediated by the test compounds and measured in thisassay can be correlated with immunosuppressive, antifungal,antiproliferative and toxicity profiles, as well as their Ki's forinhibition of FKBP PPIase activity.

Using the quantitative chemiluminescence assay described above, theinteraction of human LexA-FKBP12 and VP16-RAPT1 was analyzed in thepresence and absence of rapamycin. Interaction between FKBP12- and RAPT1was measured as a function of drug concentration. Addition of rapamycinfrom 0 to 500 ng/ml increased β-galactosidase activity approximately onethousand-fold. This effect was specific for rapamycin; FK506 over thesame concentration range did not increase β-galactosidase activitysignificantly over background levels. If lexA-da, a control construct,is substituted for the lexA-FKBP12, β-galactosidase activity does notincrease as a function of rapamycin addition. The basal levels ofβ-galactosidase in the negative controls are 0.1 percent of the maximumlevels detected in the yeast strain containing the FKBP12 and RAPT1constructs, grown in media containing 500 ng/ml rapamycin. Theseresults, illustrated in FIG. 2, indicate that protein interactionsmediated by a small molecule in the two-hybrid system can be quantitatedand assayed in a microtiter format that can be used for high throughputscreening. Employing various FKBPs and RAPT1 proteins in the two-hybridformat (FIG. 3) rapamycin-mediated interactions were measured in thisquantitative assay.

Example 9 In Vitro Protein Interactions Mediated by Rapamycin

Drug-mediated interactions of FK506-binding proteins and the RAPT1proteins is analyzed in vitro using purified FKBP12 fused toglutathione-S-transferase (GST) and ³⁵S labeled RAPT1 proteins preparedby in vitro transcription and translation. For this purpose FKBP12 isfused in the frame of GST in pGEX (Pharmacia, Piscataway, N.J.).GST-FKBP12 fusion proteins are expressed and purified from E. coli(Vojtek et al. (1993) Cell 74:205-214). RAPT1 coding sequences arecloned behind the CMV and T7 promoters in the mammalian expressionvector, pX (Superti-Furga et al. (1991) J. Immunol. Meths. 151:237-244).RAPT1 sequences are transcribed from the T7 promoter and translated invitro using commercially available reagents (Promega, Madison, Wis.) ina reaction containing ³⁵S-methionine. For in vitro binding (Toyoshima etal. (1994) Cell 78:67-74), 5 to 20 μA of the in vitrotranscription/translation reactions are added to 200 μl of bindingbuffer (20 mM HEPES[pH7.4], 150 mM NaCl, 10% glycerol, 0.05% NP-40).After addition of 10 μl of GST-FKBP12 bound to glutathione-agarosebeads, the reaction is incubated at 4° C. for 2 hr with rotation.Various contractions of drug are added to reactions, such as 0.1 to10-fold that of FKBP12 on a molar basis. No drug is added to controlreactions. The agarose beads are then precipitated and washed four timeswith binding buffer. Bound proteins isolated by boiling in Laemmlisample buffer, resolved on 4-20% gradient SDS polyacrylamide gels, andvisualized by autoradiography. Detection of ³⁵S-labelled RAPT1 proteinfrom binding reactions containing drug demonstrates direct binding toFKBP12 as a function of drug.

Example 10 Effect of RAPT1 Mutations on Complex Formation and RapamycinSensitivity

To more particularly map the rapamycin-binding domain of RAPT1 requiresthe isolation of mutants that fail to bind to a FKBP/rapamycin complex.As described in the Examples above, association with the FKBP/rapamycincan be tested in the LexA two-hybrid system in which FKBP12 is expressedas a fusion to LexA and RAPT1 proteins are expressed as fusions to theVP16 activation domain. Accordingly, a library of mutant RAPT1 proteinsis generated by mutagenizing coding sequences through PCR-generatedrandom mutagenesis (Cadwell and Joyce (1992) PCR Methods Appl 2:28-33).The 5′ and 3′ oligos for PCR contain BamH1 and EcoRI restriction sites,respectively, that allow subsequent cloning of the PCR products intopVP16 creating an in-frame fusion. In addition, the 3′ oligo contains a27 bp HA epitope sequence followed by an in frame stop codon. Theaddition of the HA epitope tag to the C-terminal end of the fusionproteins allows the characterization of the mutant RAPT1 proteins (seebelow).

Upon completion of the mutagenesis, the EcoR1-BamHI digested PCRproducts are inserted into pVP16. The library of mutant RAPT1 proteinsis amplified by transformation into E. coli. To identify those mutationsthat impair the ability of a RAPT1 to interact with an FKBP/rapamycincomplex, the mutagenized RAPT1 library is introduced into a yeast straincontaining the LexA-FKBP bait protein. Each transformed cell carries oneindividual mutant RAPT1 fused to the transcriptional activator VP16.Interaction between the FKBP and wild type RAPT1 occurs when cells aregrown in media containing rapamycin, inducing lacZ expression andturning colonies blue on X-GAL indicator plates. Colonies in which theinteraction between an FKBP/rapamycin complex and the RAPT1 mutant isimpaired are light blue or white. Two classes of mutations can producethis phenotype: nonsense mutations resulting in truncated version ofRAPT1 or sense mutations that affect the binding of RAPT1 to theFKBP/rapamycin complex. To distinguish between these two types ofmutations, total protein extracts made from these colonies is subjectedto Western blot analysis using an anti-HA antibody. Nonsense mutationsthat give rise to shorter, truncated proteins do not contain the HAepitope at their C-terminus and thus are not be detected by the anti-HAantibody. Conversely, full-length proteins with an incorporated sensemutations are detected with this antibody.

The library plasmids from the light blue or white colonies that expressfull-length RAPT1 protein with the HA epitope are rescued byretransformation into E Coli. The position of the mutation is determinedby sequence analysis, and the phenotype verified by retransformation ofthese plasmids back into the yeast strain containing LexA-FKBP12.

Mutants that retest can also be cloned into the mammalian expressionvector, pX. pX-RAPT1 or pX lacking RAPT1 sequences, are then introducedinto the lymphoid (CTLL and Kit225) and nonlymphoid cells (MG63 andRH30) sensitive to rapamycin. The effect of the mutation on rapamycinsensitivity is measured in terms of inhibition of DNA synthesismonitored by BrdU incorporation. Mutants that confer resistance ofrapamycin by virtue of being unable to bind to the FKBP12/rapamycincomplex indicate which mutations mediate drug sensitivity in lymphoidand nonlymphoid cells. Of particular interest is whether differentRAPT1s mediate drug sensitivity in different cell types.

Example 11 Cloning of a RAPT1-like Polypeptide from Candida albican

In order to clone homologs of the RAPT1 genes from human pathogenCandida, degenerate oligonucleotides based on the conserved regions ofthe RAPT1 and TOR proteins were designed and used to amplify C. albicanscDNA in λZAP (strain 3153A). The amplification consisted of 30 cycles of94° C. for 1 minute, 55° C. for 1 minute and 72° C. for 1 minute withthe PCR amplimers GGNAARGCNCAYCCNCARGC and ATNGCNGGRTAYTGYTGDATNTC. ThePCR reactions were separated on a 2.5% low melting agarose gel, thatidentified a sizable fragment. The fragment was eluted and cloned intopCRII (TA cloning system, Invitrogen corporation).

The C. albicans DNA probes were ³²P-labeled by nick translation and usedon Southern blots to confirm the species identity of the fragments andto further screen C. albicans cDNA libraries. Sequencing of the largercDNAs confirmed the identity of the clones. The partial sequence of a C.albicans RAPT1-like polypeptide, with the open-reading frame designated,is provided by SEQ ID Nos. 13 and 14.

All of the above-cited references and publications are herebyincorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A soluble polypeptide which specifically binds an FKBP/rapamycincomplex, which binding is rapamycin-dependent. 2-50. (canceled)
 51. Aprobe/primer comprising a substantially purified oligonucleotide,wherein the oligonucleotide comprises a region of nucleotide sequencewhich hybridizes under stringent conditions, including a wash step of0.2×SSC at 65° C., to at least 20 consecutive nucleotides of sense orantisense sequence of SEQ ID No: 1, SEQ ID No: 11, or of the 3.74 kbgene insert of pIC524 having ATCC Accession No. 75787.